Mechanisms of cytoplasmic pH regulation in alkaliphilic strains of Bacillus

The central challenge for extremely alkaliphilic Bacillus species is the need to establish and sustain a cytoplasmic pH that is over two units lower than the highly alkaline medium. Its centrality is suggested by the strong correlation between the growth rate in the upper range of pH for growth, i.e., at values above pH 10.5, and the cytoplasmic pH. The diminishing growth rate at extremely high pH values correlates better with the rise in cytoplasmic pH than with other energetic parameters. There are also general adaptations of alkaliphiles that are crucial prerequisites for pH homeostasis as well as other cell functions, i.e., the reduced basic amino acid content of proteins or segments thereof that are exposed to the medium, and there are other challenges of alkaliphily that emerge from solution of the cytoplasmic pH problem, i.e., reduction of the chemiosmotic driving force. For cells growing on glucose, strong evidence exists for the importance of acidic cell wall components, teichuronic acid and teichuronopeptides, in alkaliphily. These wall macromolecules may provide a passive barrier to ion flux. For cells growing on fermentable carbon sources, this and other passive mechanisms may have a particularly substantial role, but for cells growing on both fermentable and nonfermentable substrates, an active Na1-dependent cycle is apparently required for alkaliphily and the alkaliphile's remarkable capacity for pH homeostasis. The active cycle involves primary establishment of an electrochemical gradient via proton extrusion, a secondary electrogenic Na⁺/H⁺ antiport to achieve net acidification of the cytoplasm relative to the outside pH, and mechanisms for Na⁺ re-entry. Recent work in several laboratories on the critical antiporters involved in this cycle has begun to clarify the number and characteristics of the porters that support active mechanisms of pH homeostasis. Received: August 1, 1997 / Accepted: August 5, 1995     

Lipid membranes from halophilic and alkali-halophilic Archaea have a low H+ and Na+ permeability at high salt concentration

The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied. In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4 M of NaCl and KCl. The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9. The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability. It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions. Received: February 25, 1999 / Accepted: June 11, 1999     

Redox-Bohr effect in electron/proton energy transduction: cytochrome c 3 coupled to hydrogenase works as a 'proton thruster' in Desulfovibrio vulgaris

 A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c ₃, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement. Received: 2 April 1997 / Accepted: 23 May 1997     

A variable stoichiometry model for pH homeostasis in bacteria.

The composition of the proton-motive force of a hypothetical bacterial cell of wide pH tolerance is analyzed according to a model whereby the electron transport chain and various proton-linked sodium and potassium ion transporting modes are responsible for the development of the membrane potential and the chemical potentials of the three cations. Simultaneous use of two or more modes employing the same metal cation, but at a different stoichiometric ratio with respect to protons, produces nonintegral stoichiometry; the modes could represent either different devices or different states of a single device. Cycling of the cation, driven by proton-motive force, results. The relative conductances of the various modes are postulated to be pH-dependent. The pattern of potentials that results is qualitatively in accord with current knowledge and may reflect the mechanism of pH homeostasis in bacteria. The membrane potential is outwardly directed (positive inside) at extremely acid pH, becoming inwardly directed as the pH increases; the pH gradient across the membrane is large and inwardly directed (alkaline inside) at acid pH, becoming smaller and eventually inverting at alkaline pH values; the transmembrane potassium gradient is outwardly directed (high concentration inside) at all pH values; the transmembrane sodium gradient is inwardly directed at all pH values, following the pH gradient from acid through neutral pH, but then diverging at alkaline pH.     

Bioenergetic properties and viability of alkalophilic Bacillus firmus RAB as a function of pH and Na+ contents of the incubation medium.

The bioenergetic properties and viability of obligately alkalophilic Bacillus firmus RAB have been examined upon incubation in alkaline and neutral buffers in the presence or absence of added Na+. At pH 10.5, cells incubated in the absence of Na+ exhibited an immediate rise in cytoplasmic pH from less than 9.5 to 10.5, and they lost viability very rapidly. Viability experiments in the presence or absence of an energy source further suggested that the Na+-dependent mechanism for pH homeostasis is an energy-requiring function. The Na+/H+ antiporter, which catalyzes the vital proton accumulation at alkaline pH, was only slightly operational at pH 7.0; both whole cells and vesicles exhibited net proton extrusion even in the presence of Na+. Moreover, cells incubated in buffer at pH 7.0 were actually more viable in the presence of Na+ than in its absence. Thus, the inability of B. firmus RAB to grow at neutral pH is not due to excessive acidification of the cytoplasm. Rather, the transmembrane electrical potential, delta psi, generated at pH 7.0 was found to be much lower than at alkaline pH. The very low delta psi compromised several cell functions, e.g., Na+/solute symport and motility, which in this and other alkalophiles specifically depend upon delta psi and Na+.     

Protonmotive force and catecholamine transport in isolated chromaffin granules.

The effect of the transmembrane potential (delta psi) and the proton concentration gradient (delta pH) across the chromaffin granule membrane upon the rate and extent of catecholamine accumulation was studied in isolated bovine chromaffin granules. Freshly isolated chromaffin granules had an intragranular pH of 5.5 as measured by [14C]methylamine distribution. The addition of ATP to a suspension of granules resulted in the generation of a membrane potential, positive inside, as measured by [14C]thiocyanate (SCN-) distribution. The addition of carboxyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a proton translocator, resulted in a reversal of the potential to negative values (measured by [3H]tetramethylphenylphosphonium (TPMP+)) approaching -90 mV. Changing the external pH of a granular suspension incubated with FCCP produced a linear perturbation in the measured potential from positive to negative values, which can be explained by the distribution of protons according to their electrochemical gradient. When ammonia (1 to 50 mM) was added to highly buffered suspensions of chromaffin granules there was a dose-dependent decrease in the transmembrane proton gradient (delta pH) and an increase in the membrane potential (delta psi). On the other hand, thiocyanate or FCCP, at varying concentration, produced a dose-related collapse of the membrane potential and had no effect upon the transmembrane proton gradient. The addition of larger concentrations of catecholamines caused a decrease in the transmembrane proton gradient and an increase in the membrane potential. Time-resolved influx of catecholamines into the granules was studied radiochemically using low external catecholamine concentrations. The accumulation of epinephrine or norepinephrine was over one order of magnitude greater in the presence of ATP than in its absence. The rate and extent of amine accumulation was found to be related to the magnitude of the membrane potential at fixed transmembrane proton concentration (delta pH) values. Likewise, the accumulation was related to the magnitude of the delta pH at fixed membrane potential values. These results suggest that the existence of both a transmembrane proton gradient and a membrane potential are required for optimal catecholamine accumulation to occur.     

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K _m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na⁺, Mg⁺⁺, Ca⁺⁺, Ni⁺⁺, Zn⁺⁺, Fe⁺⁺, EDTA and DTT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c? and K?[Fe(CN)?]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

ESR study of proton transport across phospholipid vesicle membranes

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]     

Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1

In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H. pylori were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea, H. pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H. pylori is discussed.     

Features of apparent nonchemiosmotic energization of oxidative phosphorylation by alkaliphilic Bacillus firmus OF4.

Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A. A., and Krulwich, T. A. (1989) Annu. Rev. Microbiol. 43, 435-463). To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5. As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential. The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient. Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout. When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5. The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes. Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells. A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work. A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.     

Aluminium effects on transmembrane potential in cells of fibrous roots of sugar beet

The effect of aluminium (Al) on the electrical transmembrane potential of epidermal and outer cortical root cells of intact seedlings of sugar beet (Beta vulgaris L. cv. Monohill) was studied. The potential difference to the surrounding medium was recorded with microelectrodes inserted into the vacuoles (PD_v) and cytoplasm (PD_c) of intact roots. Both long-term effects of AlCl₃ (100, μM present during cultivation) and immediate effects of AlCl₃ (10, 50, or 100 μM present in the assay medium), were measured. The effect of Al was measured at pH 4.0, 5.0 and 6.5 in order to obtain information on the toxicity of different Al forms existing at different pH values. Low pH and/or the presence of AlCl₃ during cultivation caused large depolarizations of the PD_v. Since the immediate effect of 2,4-dinitrophenol (DNP) on the resting potential of cells from Al-cultivated plants was negligible, it is likely that Al affects the metabolic component of the transmembrane potential. Aluminium also had an immediate effect on the PD in root cells of plants cultivated without Al. Addition of 10 or 50 μM Al to the assay medium caused hyperpolarization of PD_v in the presence of 0.5 mM Ca²⁺ at all pH values studied, depolarization of PD_c at pH 6.5, and hyperpolarization of PD_c at lower pH. At 1 mM Ca²⁺, or in the presence of K⁺ (≥ 2 mM), however, the same Al concentrations had little effect on PD_c. The strongest depolarizing effects of 10 or 50 μM Al in short-term treatments were obtained at pH 6.5, and were probably due to the soluble species Al(OH)₃, which is more frequent at pH 6.5 than at a lower pH. Addition of 50 μM Al caused alkalinization of the root medium at pH 6.5, but not at pH 4.0. Therefore, it is possible that Al at pH 6.5 is bound to, or translocated across, the membrane without the accompanying hydroxide ions. It is likely that most of the Al is bound to the root cells, since removal of Al from the buffer surrounding the roots did not cause the changed PD values to return to the original values. Aluminium also interacts with effects of Ca²⁺ and K⁺ on the membrane potential, since changes in PD, induced by changes in concentrations of Ca²⁺ and K⁺ are different in the absence and presence of Al.     

The proton gradient across mycoplasma membranes

The proton gradient across mycoplasma membranes was determined by using different probes which distribute between the intracellular space and the suspension medium in response to a transmembrane proton gradient. The intracellular pH of intact glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: pH_ext=7 and pH_int=7.4; hence, pH=0.4. The size of the proton gradient depended upon the extracellular pH. Without nutrient substrate, the mycoplasmas were unable to maintain a transmembrane proton gradient, i.e., pH approximated O.N, N-dicyclohexylcarbodiimide, an inhibitor of membrane-bound ATPase, carbonyl cyanide-m-chlorophenyl hydrazone, a proton conductor, and gramicidin, an antibiotic forming cation conduction channels across membranes, strongly affected and even abolished the proton gradient across mycoplasma membranes. These substances also impaired the metabolic activity and viability of mycoplasmas.     

Nonuniform lateral distribution of transmembrane ΔpH in a coupling membrane as related to its curvature

The possibility is analyzed that the pH of the water space localized inside the invagination of a membrane can differ from the pH of the external bulk buffer outside the invagination. The proton flow responsible for decreased pH values inside mitochondrial cristae and membrane invaginations of cyanobacteria has been calculated. If deltapsi (electric potential difference) inside and outside the invaginations is the same, there may exist a lateral microheterogeneity of transmembrane deltapH, and hence deltamu-H+. It seems that the invagination is a kind of buffer for accumulating deltamu-H+ in membrane systems. In eutrified waters (pH > 9) and also under the conditions of a sudden decrease or increase of light, or of a respiratory substrate of O2, ATP synthesis should proceed in the invaginated rather than in the flat regions of a membrane.     

Acid Sensitivity of a Mutant of Lactococcus lactis subsp. lactis C2 with Reduced Membrane-bound ATPase Activity

Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp. lactis C2 as spontaneous neomycin-resistant mutants. Characteristics of the representative mutant, No. 1016–51, were compared with the parental strain in cultures using a jar fermentor with the pH controlled at various values. At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical. At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24 h decreased to 60, 36, and 60% of the parental strain, respectively. During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80 h. On the other hand, at pH 4.0, the viable count of the strain No. 1016–51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability. Therefore, it was concluded that the membrane-bound ATPase is essential for this organism to survive at low pH, probably through its function of proton pumping for maintaining cytoplasmic pH levels.     

pH control of limonin debittering with entrapped Rhodococcus fascians cells

Summary Rhodococcus fascians cells were immobilized by entrapment in -carrageenan. The ability of the system to continuously degrade limonin was tested against pH. A burst of activity was observed when changing from pH 4.5 to 5.0, and a small increase could be seen above the latter value. Such behaviour was not only a response of the metabolic activity of the cells to changes in the medium pH, but to selectivity towards the chemical form of the limonin substrate, which also depends on pH. Additionally, the immobilized cells showed increased resistance against pH changes, since the system recovered almost full activity when the pH was restored to 7.0 after being operated for long periods at pH 4.0. The decrease in limonin-degrading capability of the immobilized cells at low pH values could be overcome by choosing an appropriate dilution rate.Offprint requests to: J. L. Iborra