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Redox homeostasis is the balance between oxidation and reduction reactions. Its maintenance depends on glutathione, including its reduced and oxidized form, GSH/GSSG, which is the main intracellular redox buffer, but also on the nicotinamide adenine dinucleotide phosphate, including its reduced and oxidized form, NADPH/NADP+. Under conditions that enable yeast cells to undergo fermentative metabolism, the main source of NADPH is the pentose phosphate pathway. The lack of enzymes responsible for the production of NADPH has a significant impact on yeast cells. However, cells may compensate in different ways for impairments in NADPH synthesis, and the choice of compensation strategy has several consequences for cell functioning. The present study of this issue was based on isogenic mutants: Δzwf1, Δgnd1, Δald6, and the wild strain, as well as a comprehensive panel of molecular analyses such as the level of gene expression, protein content, and enzyme activity. The obtained results indicate that yeast cells compensate for the lack of enzymes responsible for the production of cytosolic NADPH by changing the content of selected proteins and/or their enzymatic activity. In turn, the cellular strategy used to compensate for them may affect cellular efficiency, and thus, the ability to grow or sensitivity to environmental acidification. [ABSTRACT FROM AUTHOR]

Bacterial panicle blight is caused by Burkholderia glumae and results in damage to rice crops worldwide. Virulence of B. glumae requires quorum sensing (QS)‐dependent synthesis and export of toxoflavin, responsible for much of the damage to rice. The DedA family is a conserved membrane protein family found in all bacterial species. B. glumae possesses a member of the DedA family, named DbcA, which we previously showed is required for toxoflavin secretion and virulence in a rice model of infection. B. glumae secretes oxalic acid as a "common good" in a QS‐dependent manner to combat toxic alkalinization of the growth medium during the stationary phase. Here, we show that B. glumae ΔdbcA fails to secrete oxalic acid, leading to alkaline toxicity and sensitivity to divalent cations, suggesting a role for DbcA in oxalic acid secretion. B. glumae ΔdbcA accumulated less acyl‐homoserine lactone (AHL) QS signalling molecules as the bacteria entered the stationary phase, probably due to nonenzymatic inactivation of AHL at alkaline pH. Transcription of toxoflavin and oxalic acid operons was down‐regulated in ΔdbcA. Alteration of the proton motive force with sodium bicarbonate also reduced oxalic acid secretion and expression of QS‐dependent genes. Overall, the data show that DbcA is required for oxalic acid secretion in a proton motive force‐dependent manner, which is critical for QS of B. glumae. Moreover, this study supports the idea that sodium bicarbonate may serve as a chemical for treatment of bacterial panicle blight. [ABSTRACT FROM AUTHOR]

pH is a homeostatic variable tightly regulated at the cellular, tissue, and systemic levels. In particular, the regulation of tissue pH and the responses to pH perturvation remain poorly understood. Mammalian immune cells may be ideal sensors for tissue pH mediating both homeostatic and inflammatory responses. pH can be sensed by extracellular and intracellular receptors. Recent findings illustrate the crucial roles of extracellular receptors on immune cells, but the immunological functions of many pH sensing receptors remain to be uncovered. Responses to pH can be classified into two categories: effector functions that counter the pH deviation as negative feedback, and noneffector functions that facilitate adaptation to altered pH environments. Dysregulated pH detection, effector, and noneffector functions all create vulnerability for pathological outcomes. Understanding the immune mechanisms of pH-dependent response might spur the development and discovery of new therapeutic approaches in cancer and inflammatory disorders. pH deviation is often associated with pathological conditions. Novel conceptual frameworks of both homeostatic and inflammatory circuits can provide a coherent rationale for pH-dependent regulation in mammalian immune responses. These can guide mechanistic exploration of the complex relationships between microenvironment, pH sensing, immune responses, tissue homeostasis and inflammation, which will stimulate further research into understanding microenvironment-dependent immune regulation in immune-related disorders. pH is tightly maintained at cellular, tissue, and systemic levels, and altered pH – particularly in the acidic range – is associated with infection, injury, solid tumors, and physiological and pathological inflammation. However, how pH is sensed and regulated and how it influences immune responses remain poorly understood at the tissue level. Applying conceptual frameworks of homeostatic and inflammatory circuitries, we categorize cellular and tissue components engaged in pH regulation, drawing parallels from established cases in physiology. By expressing various intracellular (pHi) and extracellular pH (pHe)-sensing receptors, the immune system may integrate information on tissue and cellular states into the regulation of homeostatic and inflammatory programs. We introduce the novel concept of resistance and adaptation responses to rationalize pH-dependent immunomodulation intertwined with homeostatic equilibrium and inflammatory control. We discuss emerging challenges and opportunities in understanding the immunological roles of pH sensing, which might reveal new strategies to combat inflammation and restore tissue homeostasis. [ABSTRACT FROM AUTHOR]

Valsa mali is a destructive phytopathogenic fungus that mainly infects apple and pear trees. Infection with V. mali results in host tissue acidification via the generation of citric acid, which promote invasion. Here, two plasma membrane H+-ATPases, VmPma1 and VmPma2 , were identified in V. mali. The VmPma1 deletion mutant (∆VmPma1) displayed higher intracellular acid accumulation and a lower growth rate compared to the wild type. In contrast, the VmPma2 deletion mutant (∆VmPma2) showed no obvious phenotypic differences. Meanwhile, loss of VmPma1 , but not VmPma2 , in V. mali led to a significant decrease in growth under acidic or alkaline conditions compared with WT. More importantly, ∆VmPma1 showed a greater reduction in ATPase hydrolase activity and acidification of the external environment, more sensitivity to abiotic stress, and weaker pathogenicity than ∆VmPma2. This evidence indicates that VmPma1 is the main gene of the two plasma membrane H+-ATPases. Transcriptomic analysis indicated that many metabolic processes regulated by VmPma1 are strictly pH-regulated. Besides, we identified two genes (named VmAgn1p and Vmap1) that contribute to the pathogenicity of V. mali by differentially regulating external acidification capacity. Overall, our findings show that VmPma1 plays a pivotal role in pathogenicity by affecting the acidification of V. mali. [ABSTRACT FROM AUTHOR]

The fact that overexpression of the yeast Ser/Thr protein phosphatase Ppz1 induces a dramatic halt in cell proliferation was known long ago, but only work in the last few years has provided insight into the molecular basis for this toxicity. Overexpression of Ppz1 causes abundant changes in gene expression and modifies the phosphorylation state of more than 150 proteins, including key signaling protein kinases such as Hog1 or Snf1. Diverse cellular processes are altered: halt in translation, failure to properly adapt to low glucose supply, acidification of the cytosol, or depletion of intracellular potassium content are a few examples. Therefore, the toxicity derived from an excess of Ppz1 appears to be multifactorial, the characteristic cell growth blockage thus arising from the combination of various altered processes. Notably, overexpression of the Ppz1 regulatory subunit Hal3 fully counteracts the toxic effects of the phosphatase, and this process involves intracellular relocation of the phosphatase to internal membranes. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum is generally regarded as a safe microorganism, and widely used in the large-scale production of various amino acids and organic acids, such as l-glutamate, l-lysine and succinic acid. During the process of industrial fermentation, C. glutamicum is usually exposed to varying environmental stresses, such as variations in pH, salinity, temperature, and osmolality. Among them, pH fluctuations are regarded as one of the most frequent environmental stresses in microbial fermentation. In this review, we summarize the current knowledge of pH homeostasis mechanisms adopted by C. glutamicum for coping with low acidic pH and high alkaline pH stresses. Facing with low pH environments, C. glutamicum develops a variety of strategies to maintain intracellular pH homeostasis, such as lowering intracellular reactive oxygen species, the improvement of potassium transport, the regulation of mycothiol-related pathways, as well as the repression of sulfur assimilation. While during alkaline pH stresses, the Mrp-type Na+/H+ antiporters are shown to play a dominant role in conferring C. glutamicum cells resistance to alkaline pH. Furthermore, we also discuss the general strategies and prospects on metabolic engineering of C. glutamicum to improve alkaline or acid resistance. [ABSTRACT FROM AUTHOR]

Sulfur-associated enhanced biological phosphorus removal has recently been developed for the removal of biological nutrients. In this new bioprocess, the polymeric sulfur compound (poly-S) is crucial to connecting sulfur conversions and polyphosphate accumulation; however, its mechanisms are still elusive. This study investigated the role of poly-S in maintaining the system stability by operating a lab-scale reactor for 720 d and conducting batch experiments with various initial pH values. The main findings were as follows: i) intracellular poly-S increased from 30 to 95 mg S (g VSS)−1, whereas polyhydroxyalkanoates increased from 8 to 22 mg C (g VSS)−1; ii) glycogen increased from 7.5 to 12.5 mg C (g VSS)−1 during the first 520 d before decreasing; and 3) P removal could be maintained at 8–12.5 mg P (L)−1. The decrease in glycogen was likely because the accumulation of enough poly-S could replace glycogen to provide reducing power and buffer the inner pH. The results of batch tests confirmed that poly-S could adjust the intracellular protons under anaerobic conditions (pH always returned to neutral or neutral levels at the end of anaerobic phase) and provide cellular bioenergy (adenosine triphosphate, for P uptake, thereby maintaining net P removal). The predominant microbial communities were facultative denitrifying Thauera (11%), sulfide-oxidizing Thiobacillus (8%), and sulfate-reducing Desulfobacter (9%). However, the conventional polyphosphate-accumulating organisms were detected at very low abundance (e.g. Tetrasphaera at only 0.02%). Overall, poly-S could regulate intracellular protons and energy balance and reduce glycogen accumulation, keeping good biological P removal performance. • This study assessed the impact of intracellularly stored poly-S on pH homeostasis. • Intracellular poly-S and PHAs during the 700-day reactor operation. • Glycogen increased to 12 mg C (g VSS)−1 during the first 520 days before decreasing. • Biological P removal was independent of poly-S increase and glycogen decrease. • Poly-S adjusts the pH around 7 and supply energy for P removal. [ABSTRACT FROM AUTHOR]

Highlights • Proteomics highlights the central metabolism roles in acid tolerance of R. freirei. • An integrated model is proposed based on identified proteins. • Identified proteins reveals an inclination towards a catabolic state. • A crosstalk between acid and oxidative stress is suggested. Abstract Soil acidity strongly affects microbial diversity and represents a constraint to legume production. Rhizobium freirei is a common bean symbiont recognized by its tolerance to environmental stresses, including low pH. The protein expression profiles of R. freirei PRF 81T grown in pH 6.8 and 4.8 were analyzed to clarify the mechanisms responsible for acid tolerance in this species. Bacteria were grown up to exponential phase in tryptone-yeast (TY) medium at pH 6.8 and 4.8. Whole-cell protein extracts were separated by two-dimensional electrophoresis (2-DE), and spots that showed statistical difference in their relative volumes (%vol) between the treatments were selected and excised for identification by MALDI-TOF mass spectrometry. Data showed that protein synthesis was increased at pH 4.8, which consequently raises energy demand. Differential expression of membrane-associated proteins suggested an increased proton extrusion and decreased influx, while central metabolism seemed to be through inducing acid-consuming catabolic pathways and preventing fatty acid biosynthesis. Consequently, the respiratory chain was stimulated along with the production of reactive oxygen species (ROS). The broad range of metabolic pathways modulated by acidified pH endorses the adaptive response to environmental stresses as a multigenic character in R. freirei PRF 81. Moreover, our data emphasize the key role of central metabolism in acid stress tolerance. [ABSTRACT FROM AUTHOR]

Abstract Neoplastic cells display altered biosynthetic and bioenergetic machinery to support cell survival. Therefore, cancer cells optimally utilize all available fuel resources to pump their highly upregulated metabolic pathways. While glucose is the main carbon source, transformed cells also utilize other molecules, which can be utilized in metabolic pathways, designated as alternative fuels. Acetate is one of such alternative metabolic fuels, which is mainly consumed in carbohydrate and lipid metabolism. However, studies demonstrate the contradictory effects of acetate on tumor cell survival. Moreover, the mechanisms of its antitumor actions remain poorly understood. Further, the spectrum of acetate susceptible tumor targets needs to be characterized in order to optimize the use of acetate in maneuvering tumor progression as a therapeutic strategy. As the effect of acetate on survival properties of the tumor cells of thymic origin is not worked out, in the present study the effect of acetate was investigated against tumor cells derived from a murine thymoma designated as Dalton's Lymphoma (DL). Acetate treatment of tumor cells inhibited tumor cell survival accompanied by induction of apoptotic cell death, associated with modulated expression of cell survival regulatory HIF1α, ROS, p53, Caspase 3, Bax and HSP70 along with the elevated level of cytosolic cytochrome c. Acetate treatment also modulated the expression of pH regulators MCT-1 and V-ATPase accompanied by altered pH homeostasis. Expression of MDR and lipid metabolism regulatory molecules was also inhibited in tumor cells upon acetate exposure. Further, pre-exposure of tumor cells to α-CHC (α-cyano-4-hydroxycinnamate), an inhibitor of MCT-1, partially abrogated the cytotoxic action of acetate. These findings shed a new light regarding the effect and mechanisms of the exogenous acetate on the biology of tumor cells of thymic origin. Highlights • In vitro exposure of tumor cells to acetate causes metabolic inhibition • Acetate treatment triggers induction of apoptosis • Acetate exposed tumor cells show modulated cell survival regulation • Acetate exposure interferes with pH homeostasis [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux. [ABSTRACT FROM AUTHOR]

Potassium and glutamate are the most abundant ions in every living cell. Whereas potassium plays a major role to keep the cellular turgor and to buffer the negative charges of the nucleic acids, the major function of glutamate is to serve as the universal amino group donor. In addition, both ions are involved in osmoprotection in bacterial cells. Here, we discuss how bacterial cells maintain the homeostasis of both ions and how adaptive evolution allows them to live even at extreme potassium limitation. Interestingly, positively charged amino acids are able to partially replace potassium, likely by buffering the negative charge of DNA. A major factor involved in the control of potassium homeostasis in Gram-positive bacteria is the essential second messenger cyclic di-AMP. This nucleotide is synthesized in response to the potassium concentration and in turn controls the expression and activity of potassium transporters. We discuss the link between the two major ions, DNA and the second messenger c-di-AMP. [ABSTRACT FROM AUTHOR]

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris , has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris , and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris , which provides an alternative target for effectively controlling this contaminant in fruit juices. [Display omitted] • Acid stress (pH 3.0, 1 h) affect amino acid metabolism of A. acidoterrestris. • Glutamate, arginine and lysine improve the acid resistance of A. acidoterrestris. • Glutamate and arginine decarboxylation maintains intracellular pH homeostasis. • Amino acids alleviate cell membrane damage and deformation caused by acid stress. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans , Micrococcus flavus , Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs. [Display omitted] • We show interrelationships between effects of several AMPs on the ΔpH, the membrane potential and ATP-synthesis of bacteria. • A novel assay allows simultaneous detection of ATP leakage from bacterial cells as well as drop of intracellular ATP-levels. •.Always use multiple different bacteria and assays when studying effects of AMPs on bacterial membranes. [ABSTRACT FROM AUTHOR]

Cigarette smoking is the greatest risk factor for lung cancer, accounting for approximately 90% of all lung cancer-related deaths. Moreover, nicotine is associated with lung cancer onset and progression. Hypoxia-inducible factor 1α (HIF-1α) is involved in the metabolic reprogramming of cancer cells and accelerates cancer progression via regulation of pH and acid–base homeostasis. Previous studies have reported that nicotine upregulates HIF-1α expression. Therefore, we hypothesized that nicotine-mediated activation of HIF-1α regulates metabolic reprogramming and pH homeostasis in non-small cell lung cancer A549 cells and could potentially play a role in the progression of lung cancer. We examined the effects of nicotine on metabolic reprogramming and intracellular pH (pH i) homeostasis, which are critical for cancer progression. A549 cells were exposed to nicotine in the absence and presence of the nicotinic acetylcholine receptor antagonist, mecamylamine (MEC). We then analyzed glycolytic stress and the activity and expression of acid-extruder proteins, including the Na+–H+ exchanger 1 (NHE1) and monocarboxylate cotransporters 1 & 4 (MCT1 and MCT4, respectively). Nicotine promoted the Warburg effect, which is associated with accelerated migration of A549 cells through the activation of nicotinic acetylcholine receptors. Furthermore, nicotine upregulated the activities and expression of acid-extruder proteins, namely NHE1 and MCT4, and facilitated glycolysis. To the best of our knowledge, this is the first study to demonstrate that nicotine plays a pivotal regulatory role in metabolic reprogramming as well as regulation of pH i homeostasis in A549 cells via activation of nicotinic acetylcholine receptors and can therefore aggravate lung cancer progression. • Nicotine promoted anaerobic glycolysis of NSCLC and enhanced the Warburg effect through nAChR. • Nicotine facilitated cancer cell migration through nAChR. • We confirmed that functional NHE and MCT are present in A549 cells. • NHE1 activity accounts for more than half of the mechanistic acid extrusion in A549 cells. • Nicotine upregulated NHE and MCT expression and activities through nAChR. [ABSTRACT FROM AUTHOR]

The voltage-gated hydrogen channel Hv1 encoded in humans by the HVCN1 gene is a highly selective proton channel that allows large fluxes of protons across biological membranes. Hv1 form functional dimers of four transmembrane spanning proteins resembling the voltage sensing domain of potassium channels. Each subunit is highly selective for protons and is controlled by changes in the transmembrane voltage and pH gradient. Hv1 is most expressed in phagocytic cells where it sustains NADPH oxidase-dependent bactericidal function and was reported to facilitate antibody production by B cells and to promote the maturation and motility of spermatocytes. Hv1 contributes to neuroinflammation following brain damage and favors cancer progression possibly by extruding protons generated during aerobic glycolysis of cancer cells. Lack of specific Hv1 inhibitors has hampered translation of this knowledge to treat immune, fertility, or malignancy diseases. In this study, we show that the genetic deletion of Hv1 delays tumor development in a mouse model of granulocytic sarcoma and report the discovery and characterization of two novel bioavailable inhibitors of Hv1 channels that we validate by orthogonal assays and electrophysiological recordings. • Cancers with HVCN1 channel mutations are associated with improved patient survival • HVCN1 editing delays the formation of PLB-985-derived chloromas in mouse xenografts • High throughput screening identifies two specific and bioavailable Hv1 inhibitors • Pharmacological inhibition of Hv1 channels is a viable anti-cancer strategy [ABSTRACT FROM AUTHOR]

Calcium is important for chloroplast, not only in its photosynthetic but also nonphotosynthetic functions. Multiple Ca 2+ /H + transporters and channels have been described and studied in the plasma membrane and organelle membranes of plant cells; however, the molecular identity and physiological roles of chloroplast Ca 2+ /H + antiporters have remained unknown. Here we report the identification and characterization of a member of the UPF0016 family, CCHA1 (a chloroplast-localized potential Ca 2+ /H + antiporter), in Arabidopsis thaliana . We observed that the ccha1 mutant plants developed pale green leaves and showed severely stunted growth along with impaired photosystem II (PSII) function. CCHA1 localizes to the chloroplasts, and the levels of the PSII core subunits and the oxygen-evolving complex were significantly decreased in the ccha1 mutants compared with the wild type. In high Ca 2+ concentrations, Arabidopsis CCHA1 partially rescued the growth defect of yeast gdt1Δ null mutant, which is defective in a Ca 2+ /H + antiporter. The ccha1 mutant plants also showed significant sensitivity to high concentrations of CaCl 2 and MnCl 2 , as well as variation in pH. Taken these results together, we propose that CCHA1 might encode a putative chloroplast-localized Ca 2+ /H + antiporter with critical functions in the regulation of PSII and in chloroplast Ca 2+ and pH homeostasis in Arabidopsis . [ABSTRACT FROM AUTHOR]

The mycobacterial enzyme Rv1284 is a member of the β-carbonic anhydrase family that is considered essential for survival of the pathogen. The active site cavity of this dimeric protein is characterized by an exceptionally small volume and harbours a catalytic zinc ion coordinated by two cysteine and one histidine residue side chains. Using the natural products polycarpine and emodin as chemical probes in crystallographic experiments and stopped-flow enzyme assays, we report that the catalytic activity can be reversibly inhibited by oxidation. Oxidative conditions lead to the removal of one of the active site cysteine residues from the coordination sphere of the catalytic metal ion by engagement in a disulfide bond with another cysteine residue close by. The subsequent loss of the metal ion, which is supported by crystallographic analysis, may thus render the protein catalytically inactive. The oxidative inhibition of Rv1284 can be reversed by exposing the protein to reducing conditions. Because the physical size of the chemical probes used in the present study substantially exceeds the active site volume, we hypothesized that these compounds exert their effects from a surface-bound location and identified Tyr120 as a critical residue for oxidative inactivation. These findings link conditions of oxidative stress to pH homeostasis of the pathogen. Because oxidative stress and acidification are defence mechanisms employed by the innate immune system of the host, we suggest that Rv1284 may be a component of the mycobacterial survival strategy. Database Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers , and . [ABSTRACT FROM AUTHOR]

Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain. [ABSTRACT FROM AUTHOR]

Preserving an optimal intracellular pH is critical for cell fitness and productivity. The pH homeostasis of the industrially important filamentous fungus Trichoderma reesei ( Hypocrea jecorina ) is largely unexplored. We analyzed the impact of growth conditions on regulation of intracellular pH of the strain Rut-C30 and the strain M106 derived from the Rut-C30 that accumulates l -galactonic acid—from provided galacturonic acid—as a consequence of l -galactonate dehydratase deletion. For live-cell measurements of intracellular pH, we used the genetically encoded ratiometric pH-sensitive fluorescent protein RaVC. Glucose and lactose, used as carbon sources, had specific effects on intracellular pH of T. reesei . The growth in lactose-containing medium extensively acidified cytosol, while intracellular pH of hyphae cultured in a medium with glucose remained at a higher level. The strain M106 maintained higher intracellular pH in the presence of d -galacturonic acid than its parental strain Rut-C30. Acidic external pH caused significant acidification of cytosol. Altogether, the pH homeostasis of T. reesei Rut-C30 strain is sensitive to extracellular pH and the degree of acidification depends on carbon source. [ABSTRACT FROM AUTHOR]

Abstract: Gene encoding for α-carbonic anhydrases (α-CAs) and their functions in fundamental metabolism and biomineralization are widely identified in mollusks. However, the transcriptional regulation of α-CA genes in response to various environmental conditions remains unknown. In the present study, we characterized a cDNA encoding for an α-CA (HcCA) from the freshwater pearl mussel Hyriopsis cumingii. The spatial and temporal expression patterns of HcCA indicate that this gene is mainly expressed in the mantle of juvenile mussels. The expression profile of HcCA under various environmental conditions reveals that the transcription of HcCA is significantly regulated by Ca2+ concentration, water temperature, pH and air exposure. Our results suggest that HcCA is a crucial target gene by which the external environmental conditions affecting shell growth and pH homeostasis of H. cumingii. [Copyright &y& Elsevier]

Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments. [ABSTRACT FROM AUTHOR]

Background Helicobacter pylori, a neutralophile, colonizes the acidic environment of the human stomach by employing acid acclimation mechanisms that regulate periplasmic and cytoplasmic pH. The regulation of urease activity is central to acid acclimation. Inactive urease apoenzyme, Ure A/ B, requires nickel for activation. Accessory proteins UreE, F, G, and H are required for nickel insertion into apoenzyme. The Exb B/ Exb D/ Ton B complex transfers energy from the inner to outer membrane, providing the driving force for nickel uptake. Therefore, the aim of this study was to determine the contribution of Exb D to pH homeostasis. Materials and Methods A nonpolar exbD knockout was constructed and survival, growth, urease activity, and membrane potential were determined in comparison with wildtype. Results Survival of the ΔexbD strain was significantly reduced at pH 3.0. Urease activity as a function of pH and UreI activation was similar to the wildtype strain, showing normal function of the proton-gated urea channel, UreI. The increase in total urease activity over time in acid seen in the wildtype strain was abolished in the ΔexbD strain, but recovered in the presence of supraphysiologic nickel concentrations, demonstrating that the effect of the ΔexbD mutant is due to loss of a necessary constant supply of nickel. In acid, ΔexbD also decreased its ability to maintain membrane potential and periplasmic buffering in the presence of urea. Conclusions ExbD is essential for maintenance of periplasmic buffering and membrane potential by transferring energy required for nickel uptake, making it a potential nonantibiotic target for H. pylori eradication. [ABSTRACT FROM AUTHOR]

Adaptation to acid stress is an important factor in the transmission of intestinal microbes. The enterobacterium Escherichia coli uses a range of physiological, metabolic, and proton-consuming acid resistance mechanisms in order to survive acid stresses as low as pH 2.0. The physiological adaptations include membrane modifications and outer membrane porins to reduce proton influx and periplasmic and cytoplasmic chaperones to manage the effects of acid damage. The metabolic acid resistance systems couple proton efflux to energy generation via select components of the electron transport chain, including cytochrome bo oxidase, NADH dehydrogenase I, NADH dehydrogenase II, and succinate dehydrogenase. Under anaerobic conditions the formate hydrogen lyase complex catalyzes conversion of cytoplasmic protons to hydrogen gas. Finally, each major proton-consuming acid resistance system has a pyridoxal-5′-phosphate-dependent amino acid decarboxylase that catalyzes proton-dependent decarboxylation of a substrate amino acid to product and CO2, and an inner membrane antiporter that exchanges external substrate for internal product. [ABSTRACT FROM AUTHOR]

Four cation/H+ exchanger (CHX) proteins localize to prevacuolar compartments and plasma membrane, and sorting of AtCHX17 to its destination is dependent on the hydrophilic C tail. Quadruple mutants show reduced seed set implying a role of K+ transport and pH homeostasis in endosome trafficking important for reproduction.The importance of sorting proteins and wall materials to their destination is critical for plant growth and development, though the machinery orchestrating membrane trafficking is poorly understood. Transporters that alter the environment across endomembrane compartments are thought to be important players. Using Escherichia coli and yeast, we previously showed that several Arabidopsis Cation/H+ eXchanger (AtCHX) members were K+ transporters with a role in pH homeostasis, though their subcellular location and biological roles in plants are unclear. Co-expression of markers with CHX16, CHX17, CHX18, or CHX19 tagged with a fluorescent protein indicated these transporters associated with plasma membrane (PM) and post-Golgi compartments. Under its native promoter, AtCHX17(1–820)–GFP localized to prevacuolar compartment (PVC) and to PM in roots. Brefeldin A diminished AtCHX17–GFP fluorescence at PM, whereas wortmannin caused formation of GFP-labeled ring-like structures, suggesting AtCHX17 trafficked among PVC, vacuole and PM. AtCHX17(1–472) lacking its carboxylic tail did not associate with PVC or PM in plant cells. Single chx17 mutant or higher-order mutants showed normal root growth and vegetative development. However, quadruple (chx16chx17chx18chx19) mutants were reduced in frequency and produced 50%–70% fewer seeds, indicating overlapping roles of several AtCHX17-related transporters in reproduction and/or seed development. Together, our results suggest that successful reproduction and seed development depend on the ability to regulate cation and pH homeostasis by AtCHX17-like transporters on membranes that traffic in the endocytic and/or secretory pathways. [ABSTRACT FROM PUBLISHER]

Maintenance of a circumneutral intracellular pH is important for any organism. Acidophilic microorganisms thrive at low pH while maintaining their intracellular pH around 6.5. However, the mechanisms contributing to acidophile pH homeostasis are not well characterized. The authors investigated the proteomic response and cytoplasmic membrane fatty acid profiles of Acidithiobacillus caldus toward three pH values: 1.1, 2.5, and 4.0. Major rearrangements were observed but lower pH elicited larger changes. Differentially expressed transcription factors suggested tight transcriptional control of pH induced genes. Enzymes involved in sulfur metabolism were up-regulated at pH 1.1 suggesting either that: (1) cells required more energy for maintenance or (2) increased metabolic activity was a specific acid stress response to export intracellular protons via 1° electron transport proton pumps. Furthermore, glutamate decarboxylase, an important enzyme in Escherichia coli acid resistance, was uniquely expressed at pH 1.1. Other proteins previously shown to be involved in neutrophilic acid response, such as spermidine synthase, PspA, and toluene tolerance protein, were differentially expressed in At. caldus but require further investigation to show a direct link to pH homeostasis. Their roles in acidophilic organisms are discussed. Active modulation of fatty acid profiles was detected and suggested a more rigid membrane at low pH. [ABSTRACT FROM AUTHOR]

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar protonpumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1⁻/⁻) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1⁻/⁻ and wild-type mice (Pa?unescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915-F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1⁻/⁻ mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1⁺/⁺ mice) with Atp6v1b1⁻/⁻ mice to generate novel EGFP-B1⁻/⁻ mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFPB1 ⁺/⁺ and EGFP-B1⁻/⁻ mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1⁻/⁻ mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated. [ABSTRACT FROM AUTHOR]

Abstract: The rapidly developing resistance of fungi to antifungal drugs is a serious health problem. Today’s drugs mainly target cell membrane composition and synthesis. Moreover, some of them have serious side effects. New antifungal drugs targeting different molecular pathways are necessary. Amiodarone, an FDA approved antiarrhythmic drug displays antifungal activity. It targets calcium and pH homeostasis. In concentrations above 25μM, it inhibits the growth of the filamentous fungi Aspergillus niger. It triggers a biphasic calcium response accompanied by a high [Ca2+]c resting level and an intracellular acidification from 7.5 to 6.0, both of which are concentration dependent. Both extracellular calcium and calcium from intracellular organelles are sources of the transient second cytosolic calcium peak, whose amplitude is 0.12μM for cells treated with 0.1mM amiodarone. In P-type ATPase deficient A. niger strains pmrAΔ and pmcAΔ, the [Ca2+]c resting level after amiodarone treatment is at least twice as high as that of the wild type, which correlates with fungal viability and hypersensitivity to amiodarone. A combination of amiodarone and amphotericin B is additive in terms of cell viability and cytosolic calcium influx. In contrast, a combination of azole drugs and amiodarone has a synergistic effect on the viability of fungi. [Copyright &y& Elsevier]

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots. [ABSTRACT FROM PUBLISHER]

Abstract: Pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase (PDH) activity and thus promotes energetic switch from mitochondrial glucose oxidation to cytoplasmic glycolysis in cancerous cells (a phenomenon known as the ‘Warburg effect’) for their energy need, which facilitates the cancer progression by resisting induction of apoptosis and promoting tumor metastasis. Thus, in the present investigation, we explored the molecular mechanisms of the tumoricidal action of dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, on cells of a murine T cell lymphoma, designated as Dalton’s lymphoma (DL). In vitro treatment of tumor cells with DCA inhibited their survival accompanied by a modulation of the biophysical composition of tumor-conditioned medium with respect to pH, glucose and lactate. DCA treatment also altered expression of HIF1-α and pH regulators: VATPase and MCT1 and production of cytokines: IL-10, IL-6 and IFN-γ. Moreover, we also observed an alteration in the expression of other apoptosis and cell survival regulatory molecules: PUMA, GLUT1, Bcl2, p53, CAD, caspase-3 and HSP70. The study discusses the role of novel molecular mechanisms underlying DCA-dependent inhibition of tumor cell survival. This study shows for the first time that DCA-dependent alteration of tumor cell survival involves altered pH homeostasis and glucose metabolism. Thus, these findings will provide a new insight for therapeutic applications of DCA as a novel antineoplastic agent against T cell lymphoma. [Copyright &y& Elsevier]

Abstract: Directed cell-to-cell movement of the plant growth hormone auxin is often referred to as polar auxin transport, and has gained much interest since its discovery at the beginning of the 20th century, both by biologists and theoreticians. Computational modelling of auxin transport at tissue and whole plant scales has given valuable insights into the feedback dynamics between auxin and its transport, which often leads to cell polarisation. However, one cellular feedback mechanism that has been overlooked so far in previous models is the interplay between auxin and pH during auxin transport, even though this is well known from biology. We propose a kinetic model of such a feedback mechanism, linking knowledge about auxin-induced acidification of cell wall compartments to the chemiosmotic hypothesis of auxin transport. Our results suggest that proton fluxes may play a significant role in auxin transport. Since active auxin transport relies on the proton motive force over the cellular membrane, allocation of auxin is linked to its effects on compartmental pH. Our auxin/pH feedback model predicts enhanced accumulation of auxin in cells and increases in both auxin influx and efflux when this feedback is in effect. These results were robust in all simulations and consistent with biological evidence, thus providing a framework for generating and testing hypotheses of auxin-related polarisation events at a cellular level. [Copyright &y& Elsevier]

Candida dubliniensis, an emerging fungal pathogen, is the closest known species to the established pathogenic species Candida albicans. Despite the fact that these two species share>80% genome sequence identity, they exhibit distinct properties such as less hyphal growth, reduced pathogenicity and increased sensitivity to sodium stress and elevated temperatures in C. dubliniensis compared with C. albicans. It is, however, largely unknown whether signaling pathways are conserved in the two Candida species. Calcineurin signaling is known to be required for hyphal growth in Cryptococcus neoformans and Aspergillus fumigatus but remains elusive in C. albicans. Our recent study showed that calcineurin plays a clearly demonstrable role in controlling hyphal growth, drug tolerance and virulence in C. dubliniensis. Here, we extend our studies and show that calcineurin is conserved in controlling endoplasmic reticulum stress but distinct in governing pH homeostasis. Furthermore, we demonstrate that azole or echinocandin drugs in combination with the calcineurin inhibitor FK506 exhibit a synergistic effect against C. dubliniensis wild-type and echinocandin-resistant strains. The involvement of calcineurin in a variety of fungal virulence attributes and as a target for fungicidal synergism with azoles and echinocandins highlights the potential of combination therapy with calcineurin inhibitors for treating Candida infections. [ABSTRACT FROM AUTHOR]

Abstract: One of the best studied Salmonella spp. adaptive responses is the Acid Tolerance Response (ATR), responsible for bacterial survival under extreme acid conditions, and well characterized for Salmonella enterica serovar Typhimurium. This review covers the numerous studies carried out in the last two decades describing the influence of different environmental parameters on the S. Typhimurium ATR, including the complex molecular mechanisms implicated and involvement of the ATR in virulence. The review also deals with the food safety implications of this adaptive response. Finally, the limited data available regarding the ATR of other Salmonella enterica serovars is also discussed. [Copyright &y& Elsevier]

Abstract: Metal ion uptake is crucial for all living cells and an essential part of cellular bioenergetic homeostasis. In this study the uptake and the impact of the most abundant internal cation, potassium, were investigated in Actinobacteria, a group of high G+C Gram-positives with a number of prominent biotechnologically and medically important members. Genome analyses revealed a variety of different potassium uptake systems in this monophyletic group ranging from potassium channels common in virtually all Actinobacteria to different active carriers that were present predominantly in pathogenic members able to cope with various stress conditions. By applying Corynebacterium glutamicum as model system we provide experimental evidence that under optimal conditions a potassium channel is sufficient in bacteria for the maintenance of internal pH and membrane potential ensuring survival of cells under stress conditions. Under potassium limitation, however, viability of C. glutamicum was increased under acidic stress or during desiccation when a functional KtrAB potassium transporter from the pathogen Corynebacterium jeikeium was heterologously expressed. We provide experimental evidence that the KtrAB mediated enhanced potassium accumulation improved maintenance of internal pH and membrane potential. The results indicate that the occurrence of active potassium transport systems correlates with an improved potassium-dependent bioenergetic homeostasis and survival of bacterial cells under stress conditions. [Copyright &y& Elsevier]

The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of intracellular organelles and for the pH regulation of extracellular compartments. Because of the potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected subunits ATP6V1A (the 70-kDa "A" subunit) and ATP6V1E1 (the ubiquitous 31-kDa "E" subunit isoform) in epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both isoforms of the 56-kDa B subunit, ATP6V1B1 ("B1," typically expressed in epithelia specialized in regulated transepithelial proton transport) and ATP6V1B2 ("B2") in the OE. B1 localizes to the microvilli of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for signal transduction in CO2 detection. [ABSTRACT FROM AUTHOR]

Abstract: The physiological role of cyclopropane fatty acids (CFAs) in acid stress resistance was studied by in situ microelectrode H+ flux measurements of Escherichia coli Frag1 and its isogenic CFA-deficient mutant JBM 1. After exposure to pH 4 for 16h, net H+ influx in JBM 1 was twice that of the parent strain. H+ flux stabilisation at pH 6 after acid stress took more time in the cfa − mutant. The data suggest increased proton permeability and decreased ability to extrude H+ in the absence of CFA, and they support the hypothesis that CFAs protect E. coli in acidic environments by decreasing membrane permeability to H+. [Copyright &y& Elsevier]

The ionic speciation of sulfonamides is pH-driven and this may be crucial for their bioavailability and sorption to soil constituents, as well as for their uptake into bacterial cells. The inhibition behaviour of a bacterial test strain (Pseudomonas aeruginosa; DSM 1117), which was grown in the presence of different concentrations of 8 sulfonamides at pH values from 5 to 8, could be predicted by models that take the speciation of sulfonamides in- and outside of bacterial cells into account. Assuming a pH of 7.5 inside the cells (pH homeostasis), the strongest inhibition was predicted for the lowest external pH and for sulfonamides with the lowest pK a values. Growth experiments with Ps. aeruginosa basically reflected this predicted behaviour. However, Pantoea agglomerans – a bacterial strain isolated from arable soil – behaved surprisingly different regarding its pH dependency: all sulfonamides showed the strongest effects at pH 7 to 8 instead of being most effective at lowest pH, although the pK a dependencies followed the same pattern. Experimental and modeling results could be brought into good agreement for P. agglomerans if the cell-internal pH was admitted to approximate the external pH instead of implying pH homeostasis for modeling calculations. Thus, besides the actual concentration of sulfonamides, the pH dependent mode of reaction of different bacteria to sulfonamides may additionally govern the population dynamics in soils. [Copyright &y& Elsevier]

The responses of larval Aedes aegypti to media of pH 4,7 and 11 provide evidence for pH regulatory strategies. Drinking rates in pH 4 media were elevated 3- to 5-fold above those observed in pH 7 or 11. Total body water was elevated during acute exposure to acidic media. During chronic exposure, total body water was decreased and Malpighian tubule mitochondrial luminosity, quantified using Mitotracker Green FM, increased. Malpighian tubule secretion rates and energy demands thus appear to increase dramatically during acid exposure. In alkaline media, drinking rates were quite low. Larvae in pH 11 media excreted net acid (0.12 nequiv H+ g-1 h-1) and the pH indicators azolitmin and bromothymol blue revealed that the rectal lumen is acidic in vivo at all ambient pH values. The anal papillae (AP) were found to be highly permeant to acid-base equivalents. Ambient pH influenced the length, and the mass-specific length, of the AP in the presence of NaCl (59.9 mmol l-1). In contrast, the length and mass-specific length of AP were not influenced by ambient pH in low NaCl conditions. Mitochondrial luminosity was reduced in AP of larvae reared in acidic media, and was not elevated in alkaline media, relative to that of larvae reared in neutral media. These data suggest that the AP may compromise acid-base balance in acidic media, and may also be an important site of trade-offs between H+ homeostasis and NaCl uptake in dilute, acidic media. [ABSTRACT FROM AUTHOR]

Mice deficient in the ATP6V1B1 ("B 1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B 1-/- mice than in B 1+1+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B 1-1- mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B 1 - deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B 1 - deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-ATPase function is preserved in medullary ICs from B 1 -/- mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B 1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions. [ABSTRACT FROM AUTHOR]

Abstract: To adapt to environmental changes, plant cells very likely possess a biochemical system, using vacuoles, for maintaining cytoplasmic pH homeostasis. A simple approach is to estimate the active H+ influx and H+ efflux of isolated vacuolar vesicles, although there is no good mathematical model to describe H+ flux. To establish a new quantitative model, vacuolar vesicles were isolated from hypocotyls of mung bean (Vigna radiata L.), and pyrophosphate (PPi)- or ATP-dependent acidification was monitored using acridine orange. The change of pH inside the vesicles (pHin) was calculated using a pH calibration curve relating fluorescence quenching with ΔpH. After formation of a steady state ΔpH, passive H+ efflux was monitored after terminating pumping with ethylenediaminetetraacetate, and the relative H+ permeability coefficient (p H+) was calculated. The H+ efflux simulated using the p H+ corresponded to the H+ efflux determined experimentally. H+ influx was then calculated by subtracting the predicted H+ efflux from the experimental net H+ influx. H+ influx into vesicles driven by H+-PPase or H+-ATPase decreased exponentially as the intravesicular pHin decreased, suggesting modulation of pumping by ΔpH, pHin, or both. Finally, the PPi- or ATP-dependent H+ accumulation determined experimentally was closely simulated by the predicted H+ influx and H+ efflux. The ability to predict H+ flux under different conditions provides a powerful tool for studying pH homeostasis. [Copyright &y& Elsevier]

The effects of exogenous phytohormones (IAA, ABA, and GA3) on the intracellular (cytoplasmic) pH (pHc) in ungerminating and germinating petunia ( Petunia hybrida L.) pollen grains were studied. The pHc values were measured with fluorescein diacetate. In ungerminating pollen grains, all phytohormones reduced pHc relatively rapidly; after 10–15 min, initial value was restored. In germinating pollen grains, IAA and ABA induced a relatively rapid cytosol alkalization, which was not reversed during experiment. GA3 acidified the cytosol, i.e., exerted the effect similar to that in ungerminating pollen grains. Sodium orthovanadate suppressed completely the hormone-induced pHc shift toward alkaline values in germinating pollen grains, whereas this inhibitor did not affect pHc in the absence of phytohormones. Sodium orthovanadate also slowed the recovery of pHc after hormone-induced cytoplasm acidification in ungerminating pollen grains, reduced pHc in control ungerminating grains, and weakened substantially the effects of all phytohormones on these pollen grains. On the basis of these results, we suggested that physiological activities of phytohormones in this system were mediated by pHc modulation, namely, a transient disturbance in the cytosolic pH homeostasis, which could trigger further phytohormone-induced cell responses. We concluded that a hormone-induced cytoplasm alkalization in pollen grains was mediated by the activity of their plasma membrane H+-ATPase and that this proton pump was involved in the control of pHc in both germinating and ungerminating pollen grains. [ABSTRACT FROM AUTHOR]

Summary: Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and ecological balance of oral microbial populations. Actinomyces naeslundii is an important ureolytic organism in the oral cavity. In this study, we aimed to investigate the substrate affinity and pH optimum for ureolysis of A. naeslundii (ATCC12104), and expression of urease under different environmental factors. In addition, in vitro acid killing and pH drop experiments were used to detect the role of ureolysis in bacterial aciduricity and capacity to modulate pH homeostasis. We observed the K s value of the ureolytic activity was 7.5mM and a pH optimum near 6.5. Urease expression by A. naeslundii (ATCC12104) was affected by multiple factors, including environmental pH, glucose and nitrogen availability. The cells could be protected against acid killing through hydrolysis of physiologically relevant concentrations of urea. A. naeslundii (ATCC12104) demonstrated a significant capacity to temper glycolytic acidification in vitro at urea concentrations normally found in the oral cavity. [Copyright &y& Elsevier]

Abstract: Responses of Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus to a rapid change in extracellular pH (pHex) from pHex 6 to a range of concentrations down to pHex 3.0 were examined, using HCl and lactic acid (LA) as acidulants. A new fluorescent probe 5-(and-6)-carboxy-2′, 7′-dichlorofluorescein diacetate succinimidyl ester (CDCFDA-SE) was employed that enabled reliable measurements of intracellular pH (pHi) to a minimum pHi of 4.0. Changes in pHi and H+ fluxes from immobilised bacteria were measured using fluorescence ratio imaging microscopy (FRIM) and a non-invasive ion flux measuring technique (MIFE), respectively. L. innocua maintained a relatively constant pHi of 5.5–6.1 at pHex 4 and 5 via H+ extrusion. In contrast, L. delbrueckii subsp. bulgaricus progressively lowered pHi towards pHex over the entire pHex range examined. The type of acidulant used influenced pH regulation with both pHi and H+-fluxes being more severely affected by LA compared to HCl. Overall, our data demonstrated different adaptive strategies in these two bacteria. While L. innocua expels protons to maintain a constant pHi, L. delbrueckii subsp. bulgaricus allows proton entry after acidic treatment so that pHi follows pHex. [Copyright &y& Elsevier]

Abstract: Vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V-ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans. [Copyright &y& Elsevier]

Abstract: The capacity of bacteria to survive and grow at alkaline pH values is of widespread importance in the epidemiology of pathogenic bacteria, in remediation and industrial settings, as well as in marine, plant-associated and extremely alkaline ecological niches. Alkali-tolerance and alkaliphily, in turn, strongly depend upon mechanisms for alkaline pH homeostasis, as shown in pH shift experiments and growth experiments in chemostats at different external pH values. Transcriptome and proteome analyses have recently complemented physiological and genetic studies, revealing numerous adaptations that contribute to alkaline pH homeostasis. These include elevated levels of transporters and enzymes that promote proton capture and retention (e.g., the ATP synthase and monovalent cation/proton antiporters), metabolic changes that lead to increased acid production, and changes in the cell surface layers that contribute to cytoplasmic proton retention. Targeted studies over the past decade have followed up the long-recognized importance of monovalent cations in active pH homeostasis. These studies show the centrality of monovalent cation/proton antiporters in this process while microbial genomics provides information about the constellation of such antiporters in individual strains. A comprehensive phylogenetic analysis of both eukaryotic and prokaryotic genome databases has identified orthologs from bacteria to humans that allow better understanding of the specific functions and physiological roles of the antiporters. Detailed information about the properties of multiple antiporters in individual strains is starting to explain how specific monovalent cation/proton antiporters play dominant roles in alkaline pH homeostasis in cells that have several additional antiporters catalyzing ostensibly similar reactions. New insights into the pH-dependent Na+/H+ antiporter NhaA that plays an important role in Escherichia coli have recently emerged from the determination of the structure of NhaA. This review highlights the approaches, major findings and unresolved problems in alkaline pH homeostasis, focusing on the small number of well-characterized alkali-tolerant and extremely alkaliphilic bacteria. [Copyright &y& Elsevier]

Mrp systems are a novel and broadly distributed type of monovalent cation/proton antiporter of bacteria and archaea. Monovalent cation/proton antiporters are membrane transport proteins that catalyze efflux of cytoplasmic sodium, potassium or lithium ions in exchange for external hydrogen ions (protons). Other known monovalent cation antiporters are single gene products, whereas Mrp systems have been proposed to function as hetero-oligomers. A mrp operon typically has six or seven genes encoding hydrophobic proteins all of which are required for optimal Mrp-dependent sodium-resistance. There is little sequence similarity of Mrp proteins to other antiporters but three of these proteins have significant sequence similarity to membrane embedded subunits of ion-translocating electron transport complexes. Mrp antiporters have essential roles in the physiology of alkaliphilic and neutralophilic Bacillus species, nitrogen-fixing Sinorhizobium meliloti and in the pathogen Staphylococcus aureus, although these bacteria contain multiple monovalent cation/proton antiporters. The wide distribution of Mrp systems leads to the anticipation of important roles in an even wider variety of pathogens, extremophiles and environmentally important organisms. Here, the distribution, established physiological roles and catalytic activities of Mrp systems are reviewed, hypotheses regarding their complexity are discussed and major open questions about their function are highlighted. [ABSTRACT FROM AUTHOR]

The multisubunit vacuolar-type H+ATPases mediate acidification of various intracellular organelles and in some tissues mediate H+ secretion across the plasma membrane. Mutations in the B1-subunit of the apical H+ATPase that secretes protons in the distal nephron cause distal renal tubular acidosis in humans, a condition characterized by metabolic acidosis with an inappropriately alkaline urine. To examine the detailed cellular and organismal physiology resulting from this mutation, we have generated mice deficient in the B1-subunit (Atp6v1b1-/- mice). Urine pH is more alkaline and metabolic acidosis is more severe in Atp6v1b1-/- mice after oral acid challenge, demonstrating a failure of normal urinary acidification. In Atp6v1b1-/- mice, the normal urinary acidification induced by a lumen-negative potential in response to furosemide infusion is abolished. After an acute intracellular acidification, Na+-independent pH recovery rates of individual Atp6v1b1-/- intercalated cells of the cortical collecting duct are markedly reduced and show no further decrease after treatment with the selective H+ATPase inhibitor concanamycin. Apical expression of the alternative B-subunit isoform, B2, is increased in Atp6v1b1-/- medulla and colocalizes with the H+ATPase E-subunit; however, the greater severity of metabolic acidosis in Atp6v1b1-/- mice after oral acid challenge indicates that the B2-subunit cannot fully functionally compensate for the loss of B1. Our results indicate that the B1 isoform is the major B-subunit isoform that incorporates into functional, plasma membrane H+ATPases in intercalated cells of the cortical collecting duct and is required for maximal urinary acidification. [ABSTRACT FROM AUTHOR]

Mitogens control progression through the cell cycle in non-transformed cells by complex cascades of intracellular messengers, such as Ca2+ and protons, and by cell volume changes. Intracellular Ca2+ and proton concentrations are critical for linking external stimuli to proliferation, motility, apoptosis and differentiation. This review summarizes the role in cell proliferation of calcium release from intracellular stores and the Ca2+ entry through plasma membrane Ca2+ channels. In addition, the impact of intracellular pH and cell volume on cell proliferation is discussed. [ABSTRACT FROM AUTHOR]

Btn2p is a novel coiled coil cytosolic protein in Saccharomyces cerevisiae. We report that Btn2p interacts with Yif1p, a component of a protein complex at the Golgi that functions in ER to Golgi transport. Deletion of Btn2p, btn2-Δ, results in mis-localiztion of Yif1p to the vacuole. Therefore, Btn2p may have an apparent role in intracellular trafficking of proteins. Btn2p was originally identified as being up-regulated in a btn1-Δ strain, which exhibits dysregulation of vacuolar pH, and this up-regulation of Btn2p was presumed to contribute to maintaining a stable vacuolar pH [Pearce et al. Nat. Genet. 22 (1999) 55]. We propose that up-regulation of Btn2p in btn1-Δ is an indicator of altered trafficking within the cell, and as btn1-Δ serves as a model for the lysosomal storage disorder Batten disease, that altered intracellular trafficking may contribute to some of the cellular pathological hallmarks of this disease. [Copyright &y& Elsevier]

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31 P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt ) and vacuolar pH (pHvac ) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex ) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ΔpH over the cytoplasmic membrane of 6.6–6.7 was reached (pHex 0.7–0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ΔpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ΔpH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ΔpH. Nonlethal levels of the metabolic inhibitor azide (N3 – , 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31 P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and... [ABSTRACT FROM AUTHOR]

Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. This study summarizes the characterization and role of each type of ion transport and defines the main biochemical mechanisms involved in the dissolution of bone mineral during bone resorption. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H+-adenosine triphosphatase (ATPase) coupled with Cl− conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H+-ATPase and the HCO3− source for the HCO3−/Cl− exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for pH regulation in the osteoclast. The HCO3−/Cl− exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular pH. An Na+/H+ antiporter may also contribute to the recovery of intracellular pH during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic pH. Inward-rectifying K+ channels may also be involved by compensating for the external acidification due to H+ transport. These different effects of transport processes, either on bone resorption or pH homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases. [ABSTRACT FROM AUTHOR]