Cytoplasmic pH Research Articles

Plant water transport and aquaporins in oxygen-deprived environments

Oxygen deprivation commonly affects plants exposed to flooding and soil compaction. The resulting root hypoxia has an immediate effect on plant water relations and upsets water balance. Hypoxia inhibits root water transport and triggers stomatal closure. The processes contributing to the inhibition of root hydraulic conductivity and conductance (hydraulic conductivity of the whole root system) are complex and involve changes in root morphology and the functions of aquaporins. Aquaporins (AQPs) comprise a group of membrane intrinsic proteins that are responsible for the transport of water, as well as some small neutral solutes and ions. They respond to a wide range of environmental stresses including O2 deprivation, but the underlying functional mechanisms are still elusive. The aquaporin-mediated water transport is affected by the acidification of the cytoplasm and depletion of ATP that is required for aquaporin phosphorylation and membrane functions. Cytoplasmic pH, phosphorylation, and intracellular Ca2+ concentration directly control AQP gating, all of which are related to O2 deprivation. This review addresses the structural determinants that are essential for pore conformational changes in AQPs, to highlight the underlying mechanisms triggered by O2 deprivation stress. Gene expression of AQPs is modified in hypoxic plants, which may constitute an important, yet little explored, mechanism of hypoxia tolerance. In addition to water transport, AQPs may contribute to hypoxia tolerance by transporting O2, H2O2, and lactic acid. Responses of plants to O2 deprivation, and especially those that contribute to maintenance of water transport, are highly complex and entail the signals originating in roots and shoots that lead to and follow the stomatal closure. These complex responses may involve ethylene, abscisic acid, and possibly other hormonal factors and signaling molecules in ways that remain to be elucidated.

Acid‐regulated gene expression of Helicobacter pylori: Insight into acid protection and gastric colonization

The pathogen Helicobacter pylori encounters many stressors as it transits to and infects the gastric epithelium. Gastric acidity is the predominate stressor encountered by the bacterium during initial infection and establishment of persistent infection. H.pylori initiates a rapid response to acid to maintain intracellular pH and proton motive force appropriate for a neutralophile. However, acid sensing by H.pylori may also serve as a transcriptional trigger to increase the levels of other pathogenic factors needed to subvert host defenses such as acid acclimation, antioxidants, flagellar synthesis and assembly, and CagA secretion. Helicobacter pylori were acid challenged at pH 3.0, 4.5, 6.0 vs nonacidic pH for 4 hours in the presence of urea, followed by RNA-seq analysis and qPCR. Cytoplasmic pH was monitored under the same conditions. About 250 genes were induced, and an equal number were repressed at acidic pHs. Genes encoding for antioxidant proteins, flagellar structural proteins, particularly class 2 genes, T4SS/Cag-PAI, Fo F1 -ATPase, and proteins involved in acid acclimation were highly expressed at acidic pH. Cytoplasmic pH decreased from 7.8 at pHout of 8.0 to 6.0 at pHout of 3.0. These results suggest that increasing extracellular or intracellular acidity or both are detected by the bacterium and serve as a signal to initiate increased production of protective and pathogenic factors needed to counter host defenses for persistent infection. These changes are dependent on degree of acidity and time of acid exposure, triggering a coordinated response to the environment required for colonization.

Features of Cadmium absorption by plants of spring barley

The paper present result of research on the intensity of the absorption of cadmium from the soil by plants barley. The object of the study were plant spring barley varieties Peas in various stages of development. Сadmium was added to the soil (dark-grey podzolic) as a CdCl2·2,5H2O in the concentration of 3, 15 and 30 mg Cd+2 per kg of the soil. Сadmium is one of the heavy metals which occur in the environment as a result of antropogenic activities. The best solution for maintaining non-contaminated soils and plants is to remove the sources of Cd in the environment. Given that that is essentially impossible at this time, further research needs to determine how soil and plant factors affect Cd availability on polluted soils. Of all non-essential heavy metals, Cadmium (Cd) is perhaps the metal that has attracted the most attention in soil science and plant nutrition due to its potential toxicity to humans, and also its relative mobility in the soil-plant system. The Cadmium is for plants completely unnecessary element. Of particular concern for humans are the high concentrations of Cd in grain. Established that the Cadmium content in barley amended as follows: underground faction > vegetative biomass fraction > generative fraction (control and on the variant of contamination of soil in a dose 3 mg Cd+2 per kg of the soil) and underground faction > generative fraction > vegetative biomass fraction (on the variants of experience 5 and 10 maximum possible concentration of Cd+2). Сadmium absorbed most intensive barley in the flowering stage. Cadmium is characterized by subzero migratory ability in theorgans of barley. Roots are the main accumulators of this heavy metal. Every stress conditions are signal for a plant to initiate in its cells a number of different biochemical processes (e.g. changes of cytoplasm pH, changes of electrostatic potential of cell membranes, flow of ions). It is set that getting a clean straw and clean grain ispossible only on the variants of experien-ce 1 maximum possible concentration of Cd+2 and on control. The effect of сadmium and other heavy metals on plants depends not only on its form, but also on the presence and concentration of other elements and substances, the plant species and its developmental stage are also important. Several soil conditions, such as the pH value, calcium level, content of organic substances, particulaly humus etc., are decisive for the exchangeable and water soluble, available сadmium for plants.

Avenues to molecular imaging of dying cells: Focus on cancer.

Successful treatment of cancer patients requires balancing of the dose, timing, and type of therapeutic regimen. Detection of increased cell death may serve as a predictor of the eventual therapeutic success. Imaging of cell death may thus lead to early identification of treatment responders and nonresponders, and to “patient‐tailored therapy.” Cell death in organs and tissues of the human body can be visualized, using positron emission tomography or single‐photon emission computed tomography, although unsolved problems remain concerning target selection, tracer pharmacokinetics, target‐to‐nontarget ratio, and spatial and temporal resolution of the scans. Phosphatidylserine exposure by dying cells has been the most extensively studied imaging target. However, visualization of this process with radiolabeled Annexin A5 has not become routine in the clinical setting. Classification of death modes is no longer based only on cell morphology but also on biochemistry, and apoptosis is no longer found to be the preponderant mechanism of cell death after antitumor therapy, as was earlier believed. These conceptual changes have affected radiochemical efforts. Novel probes targeting changes in membrane permeability, cytoplasmic pH, mitochondrial membrane potential, or caspase activation have recently been explored. In this review, we discuss molecular changes in tumors which can be targeted to visualize cell death and we propose promising biomarkers for future exploration.

Effect of application of exogenous nitric oxide at different critical growth stages in alleviating Fe deficiency chlorosis of peanut growing in calcareous soil

ABSTRACTThis study was to investigate peanut response to application of nitric oxide (NO) at different growth stages and the effects of NO application on peanut yield and quality in calcareous soil. Sodium nitroprusside (SNP, a NO donor) solution was poured into calcareous soil at sowing, seedling, flowering, and podding stages, respectively, or at each aforesaid critical stage. Results showed that NO application increased the content of active Fe and leaf chlorophyll, which improved the photosynthesis of peanut; enhanced the ability of resistance to oxidative stress by decreased the accumulation of O2•−, H2O2, and MDA and increased the activity of antioxidant enzymes. Nitric oxide increased the content of soil available Fe and root FCR activity, which can promote peanut absorb more Fe from the calcareous soil. What's more, peanut plants may pump a large amount of H+ from root cell membrane to consume in neutralization of HCO3−, and decrease the pH in apoplast, cytoplasm, and xylem, finally balance the mineral elements (Fe, Ca, Mg, Zn, and Cu) uptake and distribution. These results indicated that NO could improve peanut growth and development, increase peanut yield and quality. Furthermore, the application of NO at sowing or seedling stage did the most obvious effect on alleviating chlorosis of peanut in calcareous soil.

Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis.

ABSTRACTStaphylococcus aureus has three types of cation/proton antiporters. The type 3 family includes two multisubunit Na+/H+ (Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane-bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 in S. aureus. Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter-deficient KNabc Escherichia coli strain. The catalytic properties of the antiporters were measured in everted (inside out) vesicles. The Mnh1 antiporter exhibited a significant exchange of Na+/H+ cations at pH 7.5. Mnh2 showed a significant exchange of both Na+/H+ and K+/H+ cations, especially at pH 8.5. Under elevated salt conditions, deletion of the mnhA1 gene resulted in a significant reduction in the growth rate of S. aureus in the range of pH 7.5 to 9. Deletion of mnhA2 had similar effects but mainly in the range of pH 8.5 to 9.5. Double deletion of mnhA1 and mnhA2 led to a severe reduction in the S. aureus growth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters in S. aureus were also assessed via their support of virulence in a mouse in vivo infection model. Deletion of the mnhA1 gene led to a major loss of S. aureus virulence in mice, while deletion of mnh2 led to no change in virulence.IMPORTANCE This study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters in S. aureus and their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+ antiport, and it plays a significant role in maintaining halotolerance at pH 7.5 while the Mnh2 antiporter has catalytic antiporter activities for Na+/H+ and K+/H+ that have roles in both osmotolerance and halotolerance in S. aureus. Study of S. aureus with a single deletion of either mnhA1 or mnhA2 was assessed in an infection model of mice. The result shows that mnhA1, but not mnhA2, plays a major role in S. aureus virulence.

Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast

The yeast Saccharomyces cerevisiae employs multiple pathways to coordinate sugar availability and metabolism. Glucose and other sugars are detected by a G protein-coupled receptor, Gpr1, as well as a pair of transporter-like proteins, Rgt2 and Snf3. When glucose is limiting, however, an ATP-driven proton pump (Pma1) is inactivated, leading to a marked decrease in cytoplasmic pH. Here we determine the relative contribution of the two sugar-sensing pathways to pH regulation. Whereas cytoplasmic pH is strongly dependent on glucose abundance and is regulated by both glucose-sensing pathways, ATP is largely unaffected and therefore cannot account for the changes in Pma1 activity. These data suggest that the pH is a second messenger of the glucose-sensing pathways. We show further that different sugars differ in their ability to control cellular acidification, in the manner of inverse agonists. We conclude that the sugar-sensing pathways act via Pma1 to invoke coordinated changes in cellular pH and metabolism. More broadly, our findings support the emerging view that cellular systems have evolved the use of pH signals as a means of adapting to environmental stresses such as those caused by hypoxia, ischemia, and diabetes.

Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation

The ability of sperm to fertilize an egg is controlled by ion channels, one of which is the pHdependent calcium channel of sperm CatSper. For CatSper to be fully activated, the cytoplasmic pH must be alkaline, which is accomplished by either proton transporters, or a faster mechanism, such as the voltage-gated proton channel Hv1. To ensure effective regulation, these channels and regulatory proteins must be tightly compartmentalized. Here, we characterize human sperm nanodomains that are comprised of Hv1, CatSper and regulatory protein ABHD2. Superresolution microscopy revealed that Hv1 forms asymmetrically positioned bilaterally distributed longitudinal lines that span the entire length of the sperm tail. Such a distribution provides a direct structural basis for the selective activation of CatSper, and subsequent flagellar rotation along the long axis that, together with hyperactivated motility, enhances sperm fertility. Indeed, Hv1 inhibition leads to a decrease in sperm rotation. Thus, sperm ion channels are organized in distinct regulatory nanodomains that control hyperactivated motility and rotation.

The Vacuolar ATPase - A Nano-scale Motor That Drives Cell Biology.

The vacuolar H+-ATPase (V-ATPase) is a ~1MDa membrane protein complex that couples the hydrolysis of cytosolic ATP to the transmembrane movement of protons. In essentially all eukaryotic cells, this acid pumping function plays critical roles in the acidification of endosomal/lysosomal compartments and hence in transport, recycling and degradative pathways. It is also important in acid extrusion across the plasma membrane of some cells, contributing to homeostatic control of cytoplasmic pH and maintenance of appropriate extracellular acidity. The complex, assembled from up to 30 individual polypeptides, operates as a molecular motor with rotary mechanics. Historically, structural inferences about the eukaryotic V-ATPase and its subunits have been made by comparison to the structures of bacterial homologues. However, more recently, we have developed a much better understanding of the complete structure of the eukaryotic complex, in particular through advances in cryo-electron microscopy. This chapter explores these recent developments, and examines what they now reveal about the catalytic mechanism of this essential proton pump and how its activity might be regulated in response to cellular signals.

NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat.

The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system's role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma.

PB-10Highly Sensitive Fluorescence Imaging of the Cytoplasmic pH Related to the Cellular Functions

PB-10Highly Sensitive Fluorescence Imaging of the Cytoplasmic pH Related to the Cellular Functions

Live Cell Imaging Reveals pH Oscillations in Saccharomyces cerevisiae During Metabolic Transitions

Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent to which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences of oscillations. In this paper, we report sustained glycolytic oscillations in single cells without the need for cyanide. Glucose addition to immobilized cells induced pH oscillations that could be imaged with fluorescent sensors. A population of cells had oscillations that were heterogeneous in frequency, start time, stop time, duration and amplitude. These changes in cytoplasmic pH were necessary and sufficient to drive changes in NADH. Oscillators had lower mitochondrial membrane potentials and budded more slowly than non-oscillators. We also uncovered a new type of oscillation during recovery from H2O2 challenge. Our data show that pH in S. cerevisiae changes over several time scales, and that imaging pH offers a new way to measure glycolytic oscillations on individual cells.

Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression.

The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC), BBA66, and some BosR (Borrelia oxidative stress regulator)-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web.

Vma3p protects cells from programmed cell death through the regulation of Hxk2p expression

In yeast, the vacuolar proton-pumping ATPase (V-ATPase) acidifies vacuoles to maintain pH of cytoplasm. Yeast cells lacking V-ATPase activity, due to a disruption of any VMA (vacuolar membrane ATPase) gene, remain viable but demonstrate growth defects. Although it has been suggested that VMA genes are critical for phospholipid biosynthesis, the link between VMA genes and phospholipid biosynthesis is still uncertain. Here, we found that cells lacking Vma3p, one of the major V-ATPase assembly genes, had a growth defect in the absence of inositol, suggesting that Vma3p is important in phospholipid biosynthesis. Through real-time PCR, we found that cells lacking Vma3p down-regulated HXK2 expression. Furthermore, acetic acid sensitivity assay showed that cells lacking Vma3p were more sensitive to acetic acid than WT cells. HXK2 encodes hexokinase 2 which can phosphorylate glucose during phospholipid biosynthesis. Since cells lacking HXK2 are sensitive to acetic acid and this is an indicator of programmed cell death, our observations suggest that Vma3p plays an important role in programmed cell death. Taken together, we have proposed a working model to describe how Vma3p protects cells against apoptosis through the regulation of HXK2 expression.

Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.

ATP fuels the removal of metabolic end-products, including H+ ions that profoundly modulate biological activities. Energetic resources in hypoxic tumor regions are constrained by low-yielding glycolysis, and any means of reducing the cost of acid extrusion, without compromising pH homeostasis, would therefore be advantageous for cancer cells. Some cancers express connexin channels that allow solute exchange between cells, and we propose that, via this route, normoxic cells supply hypoxic neighbors with acid-neutralizing HCO3- ions. This hypothesis was tested by imaging cytoplasmic pH in spheroidal tissue growths of connexin43-positive pancreatic cancer Colo357 cells during light-controlled H+ uncaging at the hypoxic core. Cytoplasmic acid retention at the core was halved in the presence of CO2/HCO3-, but this process requires a restorative HCO3- flux. The effect of CO2/HCO3- was ablated by connexin43 inhibition or knockdown. In connexin-decoupled spheroids, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an inhibitor of HCO3- uptake, had no effect on cytoplasmic [H+] in the H+-uncaging region, indicating that DIDS-sensitive transport is not an adequate pH-regulatory strategy therein. With intact connexin-coupling, acid retention at the core increased upon DIDS treatment, indicating that HCO3- ions are taken up actively by peripheral cells and then transmitted passively to cells at the hypoxic core. Thus, the energetic burden of pH regulation is offloaded from hypoxic cells onto metabolically altruistic normoxic neighbors.-Dovmark, T. H., Hulikova, A., Niederer, S. A., Vaughan-Jones, R. D., Swietach, P. Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.

Amiloride interferes with platelet- activating factor-induced respiratory burst and MMP-9 release in bovine neutrophils independent of Na+/H+ exchanger 1.

Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na+/H+ exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent.

Role of abscisic acid and ethylene in the control of water transport-driving forces in germinating petunia male gametophyte

Data on ABA involvement in osmoregulation of in vitro germinating petunia (Petunia hybrida L.) male gametophyte were obtained. Two potential targets of ABA action in a pollen tube (PT) are identified. These are represented by (1) plasma membrane (PM) H+-ATPase, electrogenic proton pump participating in PM polarization, and (2) Ca-dependent K+-channels localized in the same membrane. It was established that a stimulatory effect of ABA on electrogenic activity of H+-ATPase is mediated by the increase in free Ca2+ level in the cytosol of a PT and reactive oxygen species (ROS) generation. Based on the results obtained on the role of K+ ions in the hormonal control of water transport-driving forces in a PT, the hypothesis suggesting that ABA stimulated pollen grains (PGs) germinating and PT growth by activating K+-channels was put forward. The revealed ABA-induced shift in cytoplasmic pH (pHc) is suggested to be involved in a cascade of the events of the progamic phase of fertilization, including pH-dependent K+-channels functioning. It was established that ABA abolishes the inhibitory effects of ethylene receptors blocker, 1-methylcyclopropene (1-MCP), and blockers of ACC and ABA synthesis (aminooxyacetic acid, AOA, and fluridone, respectively) on PT germination and growth, whereas ethrel blocks the inhibitory effect of fluridone on PT growth. In stigmas pretreated with ABA and AOA before pollination, this phytohormone was found to suppress inhibitory effect of AOA on ACC synthesis in the pollen-pistil system. All these findings, taken together, led us to the conclusion that ABA is involved in petunia male gametophyte osmoregulation interacting with ethylene at the level of ACC synthesis in the progamic phase of fertilization.

In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans.

Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCECandida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C.albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C.albicans cells or in cell populations in real time through convenient and high-throughput assays.

The spoilage yeast Zygosaccharomyces bailii: Foe or friend?

Zygosaccharomyces bailii is a non-Saccharomyces budding yeast known as one of the most aggressive food spoilage microorganisms, often isolated as a contaminant during wine fermentation, as well as from many acidic, high-sugar and canned foods. The spoilage ability relies on the yeast's unique feature of tolerating the most common preservatives such as sulphite, dimethyl dicarbonate, acetic acid and sorbic acid. Therefore, many studies have focused on the description of this peculiar tolerance with the aim of developing preventative measures against Z. bailii food spoilage. These studies demonstrated the involvement of diverse molecular and physiological mechanisms in the yeast resistance, comprising detoxification of preservatives, adaptation of the cytoplasmic pH and modulation of the cell wall/membrane composition. At the same time, the described traits unveiled Z. bailii as a novel potential workhorse for industrial bioprocesses. Here we present the yeast Z. bailii starting from important aspects of its robustness and concluding with the exploitation of its potential in biotechnology. Overall, the article describes Z. bailii from different perspectives, converging in presenting it as one of the most interesting species of the Saccharomycotina subphylum. Copyright © 2017 John Wiley & Sons, Ltd.

Cpx-dependent expression of YqjA requires cations at elevated pH.

Under alkaline pH conditions, Escherichia coli must maintain a stable cytoplasmic pH of about 7.6 that is acidic relative to the environment. Bacteria employ various mechanisms to survive alkaline pH; however, membrane cation/H+ antiporters play a primary role by facilitating inward transport of protons. Escherichia coli YqjA belongs to the DedA/Tvp38 membrane protein family and, along with its paralog YghB, is required for growth at 42°C, proper cell division and antibiotic resistance. YqjA is required for viability at alkaline pH, requiring cations sodium or potassium to support growth under these conditions, suggesting it may be a transporter. We measured yqjA expression at different pHs and cation concentrations using a yqjA promoter-lacZ fusion. We found that yqjA promoter activity was highest at alkaline pH. Increased activity of the yqjA promoter required both the transcriptional regulator CpxR, in agreement with previous results, and sodium or potassium salts at alkaline pH. Extracellular cations are also required for activity of cpxP and degP promoters at alkaline pH, suggesting this is a general property of the Cpx regulon. To our knowledge, this is the first demonstration of cation-dependent expression of Cpx-regulated genes at alkaline pH.