Cytoplasmic pH Values Research Articles

Photocatalytic Carbon Dots-Triggered Pyroptosis for Whole Cancer Cell Vaccines.

Manufacturing whole cancer cell vaccines (WCCV) with both biosafety and efficacy is crucial for tumor immunotherapy. Pyroptotic cancer cells, due to their highly immunogenic properties, present a promising avenue for the development of WCCV. However, the successful development of WCCV based on pyroptotic cancer cells is yet to be accomplished. Here, a facile strategy that utilized photocatalytic carbon dots (CDs) to induce pyroptosis of cancer cells for fabricating WCCV is reported. Photocatalytic CDs are capable of generating substantial amounts of hydroxyl radicals and can effectively decrease cytoplasmic pH values under white light irradiation. This process efficiently triggers cancer cell pyroptosis through the reactive oxygen species (ROS)-mitochondria-caspase 3-gasdermin E pathway and the proton motive force-driven mitochondrial ATP synthesis pathway. Moreover, in vitro, these photocatalytic CDs-induced pyroptotic cancer cells (PCIP) can hyperactivate macrophage (M0-M1) with upregulation of major histocompatibility complex class II expression. In vivo, PCIP induced specific immune-preventive effects in melanoma and breast cancer mouse models through anticancer immune memory, demonstrating effective WCCV. This work provides novel insights for inducing cancer cell pyroptosis and bridges the gap in the fabrication of WCCV based on pyroptotic cancer cells.

Mechanistic insights into auxin-enhancing polycyclic aromatic hydrocarbon uptake by wheat roots: Evidence from in situ intracellular pH and root-surface H+ flux

Polycyclic aromatic hydrocarbons (PAHs) are a group of extremely carcinogenic organic pollutants. Our previous findings have demonstrated that plant roots actively take up PAHs through co-transport with H+ ions. Auxin serves as a pivotal regulator of plant growth and development. However, it remains unclear whether the hormone can enhance the uptake of PAHs by plant roots. Hence, the wheat root exposed to PAHs with/without auxins was set to investigate how the auxin promotes the PAHs uptake by roots. In our study, auxin could significantly enhance the uptake of PAHs after 4 h of exposure. After the addition of auxin, the root tissue cytoplasmic pH value was decreased and the H+ influx was observed, indicating that the extracellular space was alkalinized in a short time. The increased H+ influx rate enhanced the uptake of PAHs. In addition, the H+-ATPase activity was also increased, suggesting that auxin activated two distinct and antagonistic H+ flux pathways, and the H+ influx pathway was dominant. Our findings offer important information for exploring the mechanism underlying auxin regulation of PAHs uptake and the phytoremediation of PAH-contaminated soil and water.

Influence of pH value on tube formation of human dermal microvascular endothelial cells and its molecular mechanism

Influence of pH value on tube formation of human dermal microvascular endothelial cells and its molecular mechanism

Two-photon ratiometric carbon dot-based probe for real-time intracellular pH monitoring in 3D environment

• Design and development of novel pH-sensitive two-photon ratiometric carbon dot-based probe was presented. • Probe possessed remarkable thermal stability, photostability, two-photon NIR excitation capability, and biocompatibility. • Probe demonstrated superb reversibility and ratiometric response to pH changes (4–10), upon single wavelength excitation. • Precise real-time monitoring and quantification of the intracellular pH values in 2D and 3D environments was performed. Designing a two-photon ratiometric pH-sensitive nanoprobe for real time monitoring of intracellular pH in biological environment is of great importance for better understanding the pathogenesis of diseases and the design of intracellular drug delivery-based system. However, the development of such probe remains a challenge. Here, we report for the first time the design and development of the two-photon ratiometric carbon dot-based nanoprobe and demonstrate its capability to monitor and quantify the intracellular cytoplasmic pH value, in real time. The nanoprobe comprised of a fluorescent carbon dot functionalized with a pH-responsive fluorescein dye (FACD). With increasing pH, FACD exhibits a clear ratiometric change in the emission intensity ratio, with sensitivity across a wide pH range in both extracellular and intracellular compartments. FACD is non-toxic to adipose stem cells in cell imaging experimental conditions and exhibits remarkable thermal stability, photostability, and two-photon near-infrared excitation capability. Using real-time dual-channel two-photon confocal microscopy we demonstrate the great potential of FACD as an efficient nanoprobe with high accuracy for the intracellular sensing of pH in living adipose stem cells seeded on either cell-culture dishes or on a 3D printed bioactive scaffold.

Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli.

Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2–7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.

Research progress on ion channels in human glioma

Glioma is a serious threat to human health. Increasing evidences indicate that abnormal expression and activities of a number of ion channels, e.g. voltage-gated K+, Ca2+, Cl- channels, are involved in the growth, proliferation, migration and invasion of glioma cells. Ion channels may affect the occurrence and development of glioma cells through regulating the membrane potential, adjusting the cytoplasmic volume, ion concentration, and pH value. In-depth understanding the role of ion channels in formation, development and transfer process of glioma may provide new targets for glioma prevention and treatment. In this paper, the advanced studies of ion channels in glioma were briefly reviewed. Key words: Glioma; Ion channels; Abnormal expression

Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni.

Donnan effect on chloride ion distribution as a determinant of body fluid composition that allows action potentials to spread via fast sodium channels

Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts.In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl-.Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl- ions needs to be >20 times higher outside the cell.When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.

Improved ATP supply enhances acid tolerance of Candida glabrata during pyruvic acid production

A major problem in industrial fermentation of organic acids with micro-organisms is to ensure a suitable pH in the culture broth. To circumvent this problem, we investigated the effect of citrate, which is a widely used auxiliary energy co-substrate, on cell growth, organic acid production and pH homeostasis among extracellular environment, cytoplasm and vacuole, in the pyruvic acid production by Candida glabrata CCTCC M202019 under different pH conditions. Analysis of intracellular ATP regeneration, cytoplasmic and vacuolar pH values under different culture conditions points towards a relief of stress when C. glabrata is exposed to lower pH, if citrate is added. When 50 mmol l(-1) citrate was added to the culture medium, the intracellular ATP concentrations increased by 20·5% (pH 5·5), 20·4% (pH 5·0) and 39·3% (pH 4·5), and higher pH gradients among the culture broth, cell cytoplasm and vacuoles resulted. As a consequence, the cell growth and pyruvic acid production of C. glabrata CCTCC M202019 were significantly improved under pH 5·0 and 4·5. The acid tolerance of yeast can be improved by enhancing the ATP supply, which helps to maintain higher pH gradients in the system. The results presented here expand our understanding of the physiological characteristics in eukaryotic micro-organisms under low pH conditions and provide a potential route for the further improvement of organic acids production process by process optimization or metabolic engineering.

Proton Motive Force in Rhodobacter sphaeroides Under Anaerobic Conditions in the Dark

In order to examine the mediatory role of proton motive force (∆p) or proton ATPase in H₂ production by Rhodobacter sphaeroides, ∆p was determined under anaerobic conditions in the dark, and the ATPase activity has been studied in R. sphaeroides strain A-10, isolated from Arzni mineral springs in Armenia. Membrane potential (∆φ) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (∆pH) was the difference between the external and cytoplasmic pH values, and the latter was measured by 9-aminoacridine (9-AA) fluorescence changes. At pH 7.5, ∆φ was of -94 mV and the reversed ∆pH was +30 mV, resulting in ∆p of -64 mV. The addition of N,N'-dicyclohexylcarbodiimide (DCCD), the F₀F₁-ATPase inhibitor, was not affect ∆φ. It was shown that ∆φ varies nearly linearly with ΔpH, ∆φ increased from -57.1 mV at pH 6.0 to -103.8 mV at pH 8.0; it was compensated at high external pH by a reversed ∆pH, resulting in a low ∆p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.

Improved cytoplasmic pH measurements in SNARF-1 stained plant cells by image processing

Cytoplasmic pH has long been considered to act as a secondary messenger of various cellular responses by affecting the ionization state of proteins.1 In plant biology, cytoplasmic pH has traditionally been measured, especially in guard cells and as a response to plant microorganism interactions, with pH-sensitive microelectrodes.2 More recently, the development of fluorescent pH markers, such as BCECF and SNARF-1, has allowed us to monitor cytoplasmic pH without the need for electrophysiological equipment. However, because of vacuolar structures that occupy a large volume of plant cells, simple measurements of fluorescent intensities are insufficient to provide precise cytoplasmic pH values. In this addendum, we describe our improved method to monitor cytoplasmic pH in plant cells stained by SNARF-1 by image processing using a noise-reducing filter after determination of an optimal ROI size. In addition, further developments for automated region extraction are proposed.

The acid metabolism of Annona fruit during ripening

SummaryAnnona fruit ripening is associated with a high rate of reassimilation of respired CO2, and a large increase in titrable acidity mainly due to malic acid accumulation. However, little is known about the mechanisms involved in acidity changes. In this work, the kinetic properties and activities of phosphoenolpyruvate carboxylase, malate dehydrogenase and malic enzyme, implicated in the regulation of malate metabolism and by extension in cytoplasmic pH, have been analysed. Kinetic studies revealed that the Km values of NADP-malic enzyme for malate were higher than those of malate dehydrogenase for oxaloacetate (1.44 ± 0.03 mM v 0.78 ± 0.02 mM). These results together with the higher Vmax value of malate dehydrogenase (more than 26-fold) indicated more efficiency in the reaction of malate synthesis v decarboxylation. The in vitro properties of the enzymes and the fact that the activity of malate dehydrogenase is much higher than the activity of NADP-malic enzyme account for the particular high titratable acidity in cherimoyas during ripening. Our results further indicate that the balance between malic acid synthesis and consumption seems to be effectively regulated and consistent with a controlled cytoplasmic pH value, measured by 31Phosphorus nuclear magnetic resonance spectroscopy.

Intracellular pH regulation in maize root tips exposed to ammonium at high external pH.

Ammonium-induced changes in the cytoplasmic and vacuolar pH values of excised maize (Zea mays L.) root tips, measured by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy, were correlated with the ammonium content of the tissue, determined by 14N NMR. Calculations based on these measurements indicated that the pH changes observed during exposure to 10 mM ammonium for 1 h at pH 9.0, and in the recovery following the removal of the external ammonium supply, were largely determined by the influx and efflux of the weak base NH3. Carboxylate synthesis, detected by both in vivo 13C NMR and the incorporation of [14C]bicarbonate, was stimulated by the ammonium-induced alkalinization of the root tips, but the contribution that this proton-generating process made to pH regulation during and after the ammonium treatment was quantitatively insignificant. Similarly, ammonium assimilation, which was shown to occur via the proton-generating glutamine synthetase/glutamate synthase pathway using in vivo 15N NMR, was also quantitatively insignificant in comparison with the large changes in ammonium content that occurred during the ammonium treatment and subsequent recovery. The results are discussed in relation to several recent studies in which ammonium was used to perturb intracellular pH values, and it is argued (i) that a new method for probing the subcellular compartmentation of amino acids, based on an ammonium-induced alkalinization of the cytoplasm may be difficult to implement in dense heterogeneous tissues; and (ii) that observations on the apparently proton-consuming effect of ammonium assimilation in rice root hairs may actually reflect unusually rapid assimilation.

Bacterial strategies to inhabit acidic environments.

Bacteria can inhabit a wide range of environmental conditions, including extremes in pH ranging from 1 to 11. The primary strategy employed by bacteria in acidic environments is to maintain a constant cytoplasmic pH value. However, many data demonstrate that bacteria can grow under conditions in which pH values are out of the range in which cytoplasmic pH is kept constant. Based on these observations, a novel notion was proposed that bacteria have strategies to survive even if the cytoplasm is acidified by low external pH. Under these conditions, bacteria are obliged to use acid-resistant systems, implying that multiple systems having the same physiological role are operating at different cytoplasmic pH values. If this is true, it is quite likely that bacteria have genes that are induced by environmental stimuli under different pH conditions. In fact, acid-inducible genes often respond to another factor(s) besides pH. Furthermore, distinct genes might be required for growth or survival at acid pH under different environmental conditions because functions of many systems are dependent on external conditions. Systems operating at acid pH have been described to date, but numerous genes remain to be identified that function to protect bacteria from an acid challenge. Identification and analysis of these genes is critical, not only to elucidate bacterial physiology, but also to increase the understanding of bacterial pathogenesis.

Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger

31P nuclear magnetic resonance ( 31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5–10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca 2+-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min −1 compared with 4 ml min −1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (ΔpH=1.6 and 1.4, respectively).

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) Vmax of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.

Intracellular pH stability in the aquatic resurrection plant Chamaegigas intrepidus in the extreme environmental conditions that characterize its natural habitat

Chamaegigas intrepidus Dinter (syn. Lindernia intrepidus (Dinter) Oberm.) is a poikilohydric aquatic plant that lives in rock pools on granitic outcrops in Central Namibia. The pools are only filled intermittently during the summer rains, and the plants can pass through 15–20 rehydration/dehydration cycles during a single wet season. Rehydrated plants also have to cope with substantial diurnal fluctuations in the pool pH as a result of photosynthetic CO2 uptake. We have used in vivo31P NMR spectroscopy to investigate the effect of external pH and dehydration (low water potential) on intracellular pH in the roots and submerged leaves of C. intrepidus. Increasing the external pH from 6 to 10 had no effect on the steady state cytoplasmic and vacuolar pH values of submerged leaves, but caused a slight alkalinization of the root cytoplasm. Similarly dehydration with PEG‐600 at either pH 6 or pH 10 had no effect on the cytoplasmic pH of the leaves, but it did cause a small alkalinization of the leaf vacuoles at pH 10. These results imply an unusually effective regulation of intracellular pH, consistent with the adaptation of C. intrepidus to the extreme environmental conditions of its habitat. The NMR analysis also showed that dehydration had no effect on the inorganic phosphate and phosphocholine pools, and this was taken to indicate that the cell membranes were well protected from the effects of the low water potential.

PH regulation in acid-stressed leaves of pea plants grown in the presence of nitrate or ammonium salts: studies involving 31P-NMR spectroscopy and chlorophyll fluorescence

31P nuclear magnetic resonance spectroscopy was used to monitor changes of cytoplasmic and vacuolar pH values in leaf tissues from young pea plants which had been grown in hydroponic culture with either nitrate or ammonium salts as sources of nitrogen. When acid stress was applied by the addition of 15% CO 2 to air (5.1 mM CO 2 in solution), cytoplasmic pH values decreased fast by 0.5 pH units and then increased slowly without reaching the initial pH, while vacuolar pH values decreased by 0.1 pH units. Under anaerobic conditions, the cytoplasmic pH decreased by one pH unit and the vacuolar pH increased by almost 0.4 pH units. These changes were rapidly reversed when CO 2 was removed from air or, after anaerobiosis, by aeration. However, with mannose present during and after anaerobiosis, aeration failed to bring pH values back to the levels observed before anaerobiosis. Simultaneously, mannose phosphates accumulated and cytoplasmic phosphate disappeared. Since loss of phosphate decreases ATP levels, the observations suggest that ATP-dependent pumping of protons into the vacuole restored the cytoplasmic pH partially during acidification by CO 2 and fully after anaerobiosis. Photosynthesis was initially inhibited by high CO 2 and then restored indicating that protons are exported not only across the tonoplast into the vacuole but also across the chloroplast envelope into the cytosol. No large differences in pH regulation were observed in leaves of pea plants which were grown with either nitrate or ammonium salts. Apparently, retarded growth of ammonium-fertilized plants cannot be attributed to ineffective pH regulation.

Influence of alkaline buffers on cytoplasmic pH in myocardial cells exposed to metabolic acidosis

The influence of different clinically used alkaline buffers on cytoplasmic pH in normal as well as acidotic rat myocardial cells was investigated in this study by means of the fluorescent intracellular probe 2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM). It was shown that both sodium bicarbonate and Tris buffer mixture (Tribonat ®) caused a significant and dose-dependent acidification of the cytoplasm of suspended myocardial cells with normal initial intracellular pH. This decrease was followed by a slow increase during the observation period. The initial cytoplasmic pH value was more easily reached when Tris buffer mature was used. Ringer's acetate also caused a decrease of intracellular pH, but this change persisted and was further amplified during the experiment. Carbicarb in larger dosages as well as pure trometamol (Tris) caused a pronounced dose-dependent and lasting intracellular alkalinization. Intracellular acidosis was achieved by preincubating the cells in sodium acetate. Addition of sodium bicarbonate caused an initial and dose-dependent acidification of the cytoplasm followed by a slow increase to values slightly above the induced acidosis. In contrast, Tris buffer mixture showed a tendency towards an initial acidification only when larger dosages were used, and correction of the induced acidosis was possible by use of moderate to large volumes. Ringer's acetate produced a lasting and dose-dependent decrease of cytoplasmic pH, while Carbicarb and pure trometamol caused an immediate, pronounced and persistent alkalinization. Myocardial fells with low initial cytoplasmic pH due to preincubation in an acid buffer also showed an early decrease of intracellular pH alter addition of sodium bicarbonate and Tris buffer mixture. In the case of sodium bicarbonate correction of the acid-base disturbance was not achieved during the observation period, while this was accomplished by use of larger volumes of Tris buffer mixture. Carbicarb in larger volumes caused an increase in intracellular pH. The most significant and persistent increases of cytoplasmic pH was achieved by use of pure trometamol. In conclusion, the present in vitro study implies that Tris buffer mixture (Tribonat ®) is well-suited for correction of intracellular acidosis since it acts without causing a pronounced initial intracellular acidosis or a later potentially hazardous huge cytoplasmic alkalinization.

Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining ΔpH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H +-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H +-ATPase operon, 5′ and 3′ respectively. Sequence analysis indicated the presence of three inverted-repeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steady-state cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription.