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Two alkaliphilic, Gram‐stain-negative bacterial strains (MEB004T and MEB108T) were isolated from water samples collected from Lonar lake, India. The phylogenetic analysis of their 16S rRNA gene sequences showed the highest similarity to A. delamerensis DSM 18314T (98.4%), followed by A. amylolytica DSM 18337T and A. collagenimarina JCM 14267T (97.9%). The genome sizes of strains MEB004T and MEB108T were determined to be 3,858,702 and 4,029,814 bp, respectively, with genomic DNA G + C contents of 51.4 and 51.9%. Average Nucleotide Identity, DNA–DNA Hybridization and Amino Acid Identity values between strains (MEB004T and MEB108T) and A. amylolytica DSM 18337T were (82.3 and 85.5), (25.0 and 29.2) and (86.7 and 90.2%). Both novel strains produced industrially important enzymes, such as amylase, lipase, cellulase, caseinase, and chitinase at pH 10 evidenced by the genomic presence of carbohydrate-active enzymes encoding genes. Genomic analyses further identified pH tolerance genes, affirming their adaptation to alkaline Lonar Lake. Dominant fatty acids were Summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, Summed feature 3, Sum In Feature 2 and C12:0 3OH. The prevalent polar lipids included phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The major respiratory quinone was ubiquinone-8. Based on the polyphasic data, we propose the classification of strains MEB004T and MEB108T as novel species within the genus Alkalimonas assigning the names Alkalimonas mucilaginosa sp. nov. and Alkalimonas cellulosilytica sp. nov., respectively. The type strains are MEB004T (= MCC 5208T = JCM 35954T = NCIMB 15460T) and MEB108T (= MCC 5330T = JCM 35955T = NCIMB 15461T). [ABSTRACT FROM AUTHOR]

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]

This article, titled "Editorial: Sensing of and Acclimations to Environmental pH in Plants," discusses the importance of pH in plant growth and adaptation. It highlights the discovery of pH sensors in plants and their role in responding to changes in external pH. The article also explores the impact of soil pH on plant performance and the availability of nutrients. It concludes that external pH is a crucial factor in plant function and adaptation, affecting growth, defense, and development. However, the mechanisms and consequences of pH sensing in plants are not fully understood. [Extracted from the article]

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry. [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]

A novel chitin degrading alkaliphilic bacterial strain (MEB 203 T) was isolated from sediment collected from Lonar lake, India. The strain exhibited its maximum growth at a temperature of 37 °C, with an optimal pH of 10 and a NaCl concentration of 2%. 16S rRNA gene based phylogenetic tree showed that strain was closely related to Alkalihalobacterium elongatum MCC 2982 T (98.64% similarity) followed by A. alkalinitrilicum DSM 22532 T (97.84% similarity). The genome size was 4.9 Mb with DNA G + C content of 37.7%. The dDDH value between strain MEB 203 T and A. elongatum MCC 2982 T was 26.4 ± 2.4% while OrthoANI value was 82.1%. Genome analysis revealed the presence of genes responsible for L-ectoine and cation/proton antiporter which may facilitate growth of strain in alkaline-saline habitat of Lonar lake. Strain MEB 203 T was able to utilize complex sugars such as chitin, cellulose, and starch as a carbon source at alkaline conditions which was also corroborated from the genomic presence of carbohydrate active enzymes (CAZymes). It was also able to produce biotechnologically important enzymes such as lipases and proteases which were stable at pH (9–10). The bacterium is majorly composed of C15:0 iso, C16:0 iso, and C17:0 iso (> 10%) fatty acids while diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipid (PL3) were identified as the predominant polar lipids. Based on differential physiological, biochemical, and genomic features of strain MEB 203 T, a novel species Alkalihalobacterium chitinilyticum sp. nov. (Type strain MEB 203 T = MCC 3920 T = NCIMB 15407 T = JCM 35078 T) is proposed. [ABSTRACT FROM AUTHOR]

Salt and alkaline stresses often occur together, severely threatening plant growth and crop yields. Salt stress induces osmotic stress, ionic stress, and secondary stresses, such as oxidative stress. Plants under saline-alkali stress must develop suitable mechanisms for adapting to the combined stress. Sustained plant growth requires maintenance of ion and pH homeostasis. In this review, we focus on the mechanisms of ion and pH homeostasis in plant cells under salinealkali stress, including regulation of ion sensing, ion uptake, ion exclusion, ion sequestration, and ion redistribution among organs by long-distance transport. We also discuss outstanding questions in this field. [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]

Cancer cells exhibit increased glycolytic flux and adenosine triphosphate (ATP) hydrolysis. These processes increase the acidic burden on the cells through the production of lactate and protons. Nonetheless, cancer cells can maintain an alkaline intracellular pH (pHi) relative to untransformed cells, which sets the stage for optimal functioning of glycolytic enzymes, evasion of cell death, and increased proliferation and motility. Upregulation of plasma membrane transporters allows for H+ and lactate efflux; however, recent evidence suggests that the acidification of organelles can contribute to maintenance of an alkaline cytosol in cancer cells by siphoning off protons, thereby supporting tumor growth. The Golgi is such an acidic organelle, with resting pH ranging from 6.0 to 6.7. Here, we posit that the Golgi represents a "proton sink" in cancer and delineate the proton channels involved in Golgi acidification and the ion channels that influence this process. Furthermore, we discuss ion channel regulators that can affect Golgi pH and Golgi-dependent processes that may contribute to pHi homeostasis in cancer.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH. [ABSTRACT FROM AUTHOR]

An alkaliphilic, Gram-stain-positive, non-motile, rod-shaped, and spore forming bacterial strain (MEB205T) was isolated from sediment sample collected from Lonar lake, India. The strain grew optimally at pH 10, NaCl concentration of 3.0% at 37 °C. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain MEB205T belonged to the genus Halalkalibacter in the family Bacillaceae and shared the highest sequence similarity with H. okhensis Kh10-101T (98.9%) followed by H. wakoensis N-1 T (98.7%). The assembled genome of strain MEB205T has a total length of 4.8 Mb with a G + C content of 37.8%. The dDDH and OrthoANI values between strain MEB205T and H. okhensis Kh10-101 T were 29.1% and 84.3%, respectively. Furthermore, the genome analysis revealed the presence of antiporter genes (nhaA and nhaD) and L-ectoine biosynthesis gene required for survival of the strain MEB205T in alkaline-saline habitat. The major fatty acid was C15:0 anteiso, C16:0 and C15:0 iso (> 10.0%). Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. meso-diaminopimelic acid was diagnostic diamino acid for cell wall peptidoglycan. Based on the polyphasic taxonomic studies, strain MEB205T represent a novel species of the genus Halalkalibacter for which the name Halalkalibacter alkaliphilus sp. nov. (Type strain MEB205T = MCC 3863 T = JCM 34004 T = NCIMB 15406 T) is proposed. [ABSTRACT FROM AUTHOR]

ABSTRACT Alicyclobacillus acidoterrestris, which has strong acidophilic and heat-resistant properties, can cause spoilage of pasteurized acidic juice. The current study determined the physiological performance of A. acidoterrestris under acidic stress (pH 3.0) for 1 h. Metabolomic analysis was carried out to investigate the metabolic responses of A. acidoterrestris to acid stress, and integrative analysis with transcriptome data was also performed. Acid stress inhibited the growth of A. acidoterrestris and altered its metabolic profiles. In total, 63 differential metabolites, mainly enriched in amino acid metabolism, nucleotide metabolism, and energy metabolism, were identified between acid-stressed cells and the control. Integrated transcriptomic and metabolomic analysis revealed that A. acidoterrestris maintains intracellular pH (pHi) homeostasis by enhancing amino acids decarboxylation, urea hydrolysis, and energy supply, which was verified using real-time quantitative PCR and pHi measurement. Additionally, two-component systems, ABC transporters, and unsaturated fatty acid synthesis also play crucial roles in resisting acid stress. Finally, a model of the responses of A. acidoterrestris to acid stress was proposed. IMPORTANCE Fruit juice spoilage caused by A. acidoterrestris contamination has become a major concern and challenge in the food industry, and this bacterium has been suggested as a target microbe in the design of the pasteurization process. However, the response mechanisms of A. acidoterrestris to acid stress still remain unknown. In this study, integrative transcriptomic, metabolomic, and physiological approaches were used to uncover the global responses of A. acidoterrestris to acid stress for the first time. The obtained results can provide new insights into the acid stress responses of A. acidoterrestris, which will point out future possible directions for the effective control and application of A. acidoterrestris.

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH.

ABSTRACT Mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes in eukaryotes. In fungal pathogens, conserved MAPK pathways control key virulence functions such as infection-related development, invasive hyphal growth, or cell wall remodeling. Recent findings suggest that ambient pH acts as a key regulator of MAPK-mediated pathogenicity, but the underlying molecular events are unknown. Here, we found that in the fungal pathogen Fusarium oxysporum, pH controls another infection-related process, hyphal chemotropism. Using the ratiometric pH sensor pHluorin we show that fluctuations in cytosolic pH (pHc) induce rapid reprogramming of the three conserved MAPKs in F. oxysporum, and that this response is conserved in the fungal model organism Saccharomyces cerevisiae. Screening of a subset of S. cerevisiae mutants identified the sphingolipid-regulated AGC kinase Ypk1/2 as a key upstream component of pHc-modulated MAPK responses. We further show that acidification of the cytosol in F. oxysporum leads to an increase of the long-chain base (LCB) sphingolipid dihydrosphingosine (dhSph) and that exogenous addition of dhSph activates Mpk1 phosphorylation and chemotropic growth. Our results reveal a pivotal role of pHc in the regulation of MAPK signaling and suggest new ways to target fungal growth and pathogenicity. IMPORTANCE Fungal phytopathogens cause devastating losses in global agriculture. All plant-infecting fungi use conserved MAPK signaling pathways to successfully locate, enter, and colonize their hosts. In addition, many pathogens also manipulate the pH of the host tissue to increase their virulence. Here, we establish a functional link between cytosolic pH (pHc) and MAPK signaling in the control of pathogenicity in the vascular wilt fungal pathogen Fusarium oxysporum. We demonstrate that fluctuations in pHc cause rapid reprogramming of MAPK phosphorylation, which directly impacts key processes required for infection, such as hyphal chemotropism and invasive growth. Targeting pHc homeostasis and MAPK signaling can thus open new ways to combat fungal infection.

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]

In plants, cytosolic and extracellular pH homeostasis are crucial for various physiological processes, including the uptake of macronutrients and micronutrients, cell elongation, cell expansion, and enzyme activity. Proton (H+) gradients and the membrane potential are generated by a H+ pump consisting of an active primary transporter. Plasma membrane (PM) H+-ATPase, a PM-localized H+ pump, plays a pivotal role in maintaining pH homeostasis in plant cells and extracellular regions. PM H+-ATPase activity is regulated by protein abundance and by post-translational modifications. Several stimuli have been found to activate the PM H+-ATPase through phosphorylation of the penultimate threonine (Thr) of the carboxy terminus. Light- and photosynthesisinduced phosphorylation of PM H+-ATPase are conserved phenomena among various plant species. In this work, we review recent findings related to PM H+-ATPase regulation in the photosynthetic tissues of plants, focusing on its mechanisms and physiological roles. The physiological roles of photosynthesis-dependent PM H+-ATPase activation are discussed in the context of nitrate uptake and cytoplasmic streaming in leaves. [ABSTRACT FROM AUTHOR]

In plants, cytosolic and extracellular pH homeostasis are crucial for various physiological processes, including the uptake of macronutrients and micronutrients, cell elongation, cell expansion, and enzyme activity. Proton (H+) gradients and the membrane potential are generated by a H+ pump consisting of an active primary transporter. Plasma membrane (PM) H+-ATPase, a PM-localized H+ pump, plays a pivotal role in maintaining pH homeostasis in plant cells and extracellular regions. PM H+-ATPase activity is regulated by protein abundance and by post-translational modifications. Several stimuli have been found to activate the PM H+-ATPase through phosphorylation of the penultimate threonine (Thr) of the carboxy terminus. Light- and photosynthesis-induced phosphorylation of PM H+-ATPase are conserved phenomena among various plant species. In this work, we review recent findings related to PM H+-ATPase regulation in the photosynthetic tissues of plants, focusing on its mechanisms and physiological roles. The physiological roles of photosynthesis-dependent PM H+-ATPase activation are discussed in the context of nitrate uptake and cytoplasmic streaming in leaves.

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants. [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]

ABSTRACT Cytoplasmic pH in bacteria is tightly regulated by diverse active mechanisms and interconnected regulatory processes. Many processes and regulators underlying pH homeostasis have been identified via phenotypic screening of strain libraries for nongrowth at low or high pH values. Direct screens with respect to changes of the internal pH in mutant strain collections are limited by laborious methods, which include fluorescent dyes and radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule during the generation of the strain. Here, we used the pH-sensitive mCherry variant mCherryEA as a ratiometric pH biosensor. We visualized the internal pH of Escherichia coli colonies on agar plates by the use of a GelDoc imaging system. Combining this imaging technology with robot-assisted colony picking and spotting allowed us to screen and select mutants with altered internal pH values from a small transposon mutagenesis-derived E. coli library. Identification of the transposon (Tn) insertion sites in strains with altered internal pH levels revealed that the transposon was inserted into trkH (encoding a transmembrane protein of the potassium uptake system) or rssB (encoding the adaptor protein RssB, which mediates the proteolytic degradation of the general stress response regulator RpoS), two genes known to be associated with pH homeostasis and pH stress adaptation. This successful screening approach demonstrates that the pH sensor-based analysis of arrayed colonies on agar plates is a sensitive approach for the rapid identification of genes involved in pH homeostasis or pH stress adaptation in E. coli. IMPORTANCE Phenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes. For this purpose, transcription factor-based biosensors have emerged as the sensor type of choice. Here, the target stimulus initiates the activation of a response gene (e.g., a fluorescent protein), followed by transcription, translation, and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real time. To capture rapid intracellular processes adequately, fluorescent reporter proteins are extensively applied. However, these sensor types have not previously been used for phenotypic screenings. To take advantage of their properties, we established here an imaging method that allows application of a rapid ratiometric sensor protein for assessing the internal pH of colonies in a high-throughput manner.

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.

Extreme acidophiles thrive in environments rich in protons (pH values <3) and often high levels of dissolved heavy metals. They are distributed across the three domains of the Tree of Life including members of the Proteobacteria. The Acidithiobacillia class is formed by the neutrophilic genus Thermithiobacillus along with the extremely acidophilic genera Fervidacidithiobacillus , Igneacidithiobacillus , Ambacidithiobacillus , and Acidithiobacillus. Phylogenomic reconstruction revealed a division in the Acidithiobacillia class correlating with the different pH optima that suggested that the acidophilic genera evolved from an ancestral neutrophile within the Acidithiobacillia. Genes and mechanisms denominated as "first line of defense" were key to explaining the Acidithiobacillia acidophilic lifestyle including preventing proton influx that allows the cell to maintain a near-neutral cytoplasmic pH and differ from the neutrophilic Acidithiobacillia ancestors that lacked these systems. Additional differences between the neutrophilic and acidophilic Acidithiobacillia included the higher number of gene copies in the acidophilic genera coding for "second line of defense" systems that neutralize and/or expel protons from cell. Gain of genes such as hopanoid biosynthesis involved in membrane stabilization at low pH and the functional redundancy for generating an internal positive membrane potential revealed the transition from neutrophilic properties to a new acidophilic lifestyle by shaping the Acidithiobacillaceae genomic structure. The presence of a pool of accessory genes with functional redundancy provides the opportunity to "hedge bet" in rapidly changing acidic environments. Although a core of mechanisms for acid resistance was inherited vertically from an inferred neutrophilic ancestor, the majority of mechanisms, especially those potentially involved in resistance to extremely low pH, were obtained from other extreme acidophiles by horizontal gene transfer (HGT) events. [ABSTRACT FROM AUTHOR]

The voltage-gated proton channel HVCN1 is a member of the voltage-gated ion channel family. HVCN1 channel controls acid extrusion and regulates pH homeostasis in various cell types. Recent evidence indicated that the HVCN1 channel was associated with cardiac function. To investigate the role of HVCN1 in cardiac myocytes, we performed an RNA sequencing analysis of murine hearts and showed that HVCN1 null hearts exhibited a differential transcriptome profile compared with wild-type hearts. The RNA-seq data indicating impaired pH homeostasis in HVCN1 null hearts were the downregulated NADPH oxidoreductases (NOXs) and decreased expression of Cl−/HCO3− exchanger, indicating HVCN1 is a regulator of gene transcriptional networks controlling NOX signaling and CO2 homeostasis in the heart. Additionally, HVCN1 null hearts exhibited differential expression of cardiac ion channels, suggesting a potential role of HVCN1 in cardiac electrophysiological remodeling. The study highlights the importance of HVCN1 in cardiac function and may present a novel target associated with heart diseases.

Extreme acidophiles thrive in environments rich in protons (pH values

Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w. Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum. [ABSTRACT FROM AUTHOR]

Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w. Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum.

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex. [ABSTRACT FROM AUTHOR]

Cancer cells exhibit increased glycolytic flux and adenosine triphosphate (ATP) hydrolysis. These processes increase the acidic burden on the cells through the production of lactate and protons. Nonetheless, cancer cells can maintain an alkaline intracellular pH (pHi) relative to untransformed cells, which sets the stage for optimal functioning of glycolytic enzymes, evasion of cell death, and increased proliferation and motility. Upregulation of plasma membrane transporters allows for H+ and lactate efflux; however, recent evidence suggests that the acidification of organelles can contribute to maintenance of an alkaline cytosol in cancer cells by siphoning off protons, thereby supporting tumor growth. The Golgi is such an acidic organelle, with resting pH ranging from 6.0 to 6.7. Here, we posit that the Golgi represents a “proton sink” in cancer and delineate the proton channels involved in Golgi acidification and the ion channels that influence this process. Furthermore, we discuss ion channel regulators that can affect Golgi pH and Golgi-dependent processes that may contribute to pHi homeostasis in cancer.

Members of the genus Methylacidiphilum, a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5–3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h−1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation.

Methyl jasmonate (MJ) displays antineoplastic potential against numerous neoplastic cells. However, several mechanistic aspects of its antineoplastic action against malignancies of T cell origin remain elusive. The present investigation reports the novel targets of MJ and mechanistic pathways of MJ-mediated antineoplastic and chemosensitizing action against tumor cells derived from murine T-cell lymphoma, designated as Dalton’s lymphoma (DL). The present study demonstrates that MJ directly docks to HIF-1α, hexokinase 2, and Hsp70 at prominent binding sites. MJ exhibits tumoricidal action against tumor cells via induction of apoptosis and necrosis through multiple pathways, including declined mitochondrial membrane potential, enhanced expression of ROS, altered pH homeostasis, an elevated level of cytosolic cytochrome c, and modulated expression of crucial cell survival and metabolism regulatory molecules. Additionally, this study also reports the chemosensitizing ability of MJ against T cell lymphoma accompanied by a declined expression of MDR1. This study sheds new light by demonstrating the implication of novel molecular mechanisms underlying the antitumor action of MJ against T-cell lymphoma and hence has immense translational significance.

Members of the genus Methylacidiphilum , a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5–3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h−1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation. [ABSTRACT FROM AUTHOR]

ABSTRACT The ability for cells to maintain homeostasis in the presence of extracellular stress is essential for their survival. Stress adaptations are especially important for microbial pathogens to respond to rapidly changing conditions, such as those encountered during the transition from the environment to the infected host. Many fungal pathogens have acquired the ability to quickly adapt to changes in extracellular pH to promote their survival in the various microenvironments encountered during a host infection. For example, the fungus-specific Rim/Pal alkaline response pathway has been well characterized in many fungal pathogens, including Cryptococcus neoformans. However, alternative mechanisms for sensing and responding to host pH have yet to be extensively studied. Recent observations from a genetic screen suggest that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. This work explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for growth in an alkaline environment and that an elevated pH is sufficient to induce Sre1 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol but is not dependent on Rim pathway signaling, suggesting that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response and pathogenesis. IMPORTANCE The work described here further elucidates how microorganisms sense and adapt to changes in their environment to establish infections in the human host. Specifically, we uncover a novel mechanism by which an opportunistic human fungal pathogen, Cryptococcus neoformans, responds to increases in extracellular pH in order to survive and thrive within the relatively alkaline environment of the human lung. This mechanism, which is intimately linked with fungal membrane sterol homeostasis, is independent of the previously well-studied alkaline response Rim pathway. Furthermore, this ergosterol-dependent alkaline pH response is present in Candida albicans, indicating that this mechanism spans diverse fungal species. These results are also relevant for novel antimicrobial drug development as we show that currently used ergosterol-targeting antifungals are more active in alkaline environments.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells.

Abstract Background Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools. Results Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h−1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow). Conclusions Clostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells. [ABSTRACT FROM AUTHOR]

Studies with Saccharomyces cerevisiae indicated that non-physiologically high levels of acetic acid promote cellular acidification, chronological aging, and programmed cell death. In the current study, we compared the cellular lipid composition, acetic acid uptake, intracellular pH, growth, and chronological lifespan of wild-type cells and mutants lacking the protein kinase Sch9 and/or a functional V-ATPase when grown in medium supplemented with different acetic acid concentrations. Our data show that strains lacking the V-ATPase are especially more susceptible to growth arrest in the presence of high acetic acid concentrations, which is due to a slower adaptation to the acid stress. These V-ATPase mutants also displayed changes in lipid homeostasis, including alterations in their membrane lipid composition that influences the acetic acid diffusion rate and changes in sphingolipid metabolism and the sphingolipid rheostat, which is known to regulate stress tolerance and longevity of yeast cells. However, we provide evidence that the supplementation of 20 mM acetic acid has a cytoprotective and presumable hormesis effect that extends the longevity of all strains tested, including the V-ATPase compromised mutants. We also demonstrate that the long-lived sch9Δ strain itself secretes significant amounts of acetic acid during stationary phase, which in addition to its enhanced accumulation of storage lipids may underlie its increased lifespan.

The prevalence of fungal infections and their resistance patterns in fungal isolates from large number of patients with impaired immunity still remains poorly monitored. In spite of significant advances being made in the improvement of antifungal drugs, only a limited number of antifungal drugs are currently available. The present study aimed to gain further mechanistic insights into the previously described anticandidal activity of natural monoterpenoid, perillyl alcohol (PA). We found that cellular transport across cell membrane was abrogated in presence of PA. This was demonstrated by dose and time dependent enhanced cellular leakage accompanied by inhibited sodium and potassium cellular transport. In addition, we found disrupted pH homeostasis which was depicted by enhanced extracellular pH. We further observed that mitochondrial energy status is highly integrated with the antifungal activity of PA. This was evident from inhibited propidium iodide (PI) uptake in presence of sodium azide and di-nitro phenol (DNP) which showed no fluorescence when treated with PA. Moreover, we observed that PA leads to disrupted mitochondrial membrane potential. Additional cell death hallmarks in response to PA such as nuclear fragmentation was also observed with 4',6-diamidino-2-phenylindole (DAPI) staining. Taken together, PA is a novel candidate that deserves further attention to be exploited as effective antifungal agent of pharmacological interest.

Abstract Hypercapnia and subsequent respiratory acidosis are serious complications in many patients with respiratory disorders. The acute response to hypercapnia is buffering of H+ by hemoglobin and cellular proteins but this effect is limited. The chronic response is renal compensation that increases HCO3− reabsorption, and stimulates urinary excretion of titratable acids (TA) and NH4+. However, the main effective pathway is the excretion of NH4+ in the collecting duct. Our hypothesis is that, the renal NH3/NH4+ transporters, Rhbg and Rhcg, in the collecting duct mediate this response. The effect of hypercapnia on these transporters is unknown. We conducted in vivo experiments on mice subjected to chronic hypercapnia. One group breathed 8% CO2 and the other breathed normal air as control (0.04% CO2). After 3 days, the mice were euthanized and kidneys, blood, and urine samples were collected. We used immunohistochemistry and Western blot analysis to determine the effects of high CO2 on localization and expression of the Rh proteins, carbonic anhydrase IV, and pendrin. In hypercapnic animals, there was a significant increase in urinary NH4+ excretion but no change in TA. Western blot analysis showed a significant increase in cortical expression of Rhbg (43%) but not of Rhcg. Expression of CA‐IV was increased but pendrin was reduced. These data suggest that hypercapnia leads to compensatory upregulation of Rhbg that contributes to excretion of NH3/NH4+ in the kidney. These studies are the first to show a link among hypercapnia, NH4+ excretion, and Rh expression.