Ecophysiological adaptations of anaerobic bacteria to low pH: analysis of anaerobic digestion in acidic bog sediments.

The dynamics of anaerobic digestion were examined in the low-pH sediments of Crystal Bog in Wisconsin. The sediments (pH 4.9) contained 71% organic matter and the following concentrations of dissolved gases (micromoles per liter): CO2, 1,140; CH4, 490; and H2, 0.01. The rate of methane production was 6.2 mumol/liter of sediment per h, which is slower than eutrophic, neutral sediments. Microbial metabolic processes displayed the following pH optima: hydrolysis reactions, between 4.2 and 5.6; aceticlastic methanogenesis, 5.2; and hydrogen-consuming reactions, 5.6. The turnover rate constants for key intermediary metabolites were (h-1): glucose, 1.10; lactate, 0.277; acetate, 0.118; and ethanol, 0.089. The populations of anaerobes were low, with hydrolytic groups (10(6)/ml) several orders of magnitude higher than methanogens (10(2)/ml). The addition of carbon electron donors to the sediment resulted in the accumulation of hydrogen, whereas the addition of hydrogen resulted in the accumulation of fatty acids and the inhibition of hydrogen-producing acetogenic reactions. Strains of Lactobacillus, Clostridium, and Sarcina ventriculi were isolated from the bog, and their physiological attributes were characterized in relation to hydrolytic process functions in the sediments. The present studies provide evidence that the pH present in the bog sediments alter anaerobic digestion processes so that total biocatalytic activity is lower but the general carbon and electron flow pathways are similar to those of neutral anoxic sediments.     

Influence of pH extremes on sporulation and ultrastructure of Sarcina ventriculi.

Distinct morphological changes in the ultrastructure of Sarcina ventriculi were observed when cells were grown in medium of constant composition at pH extremes of 3.0 and 8.0. Transmission electron microscopy revealed that at low pH (less than or equal to 3.0) the cells formed regular packets and cell division was uniform. When the pH was increased (to greater than or equal to 7.0), the cells became larger and cell division resulted in irregular cells that varied in shape and size. Sporulation occurred at high pH (i.e., greater than or equal to 8.0). The sporulation cycle followed the conventional sequence of development for refractile endospores, with the appearance of a cortex and multiple wall layers. The spores were resistant to oxygen, lysozyme, or heating at 90 degrees C for 15 min. Spores germinated within the pH range of 4.6 to 7.0.     

Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes.

The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.     

Generation of a large, protonophore-sensitive proton motive force and pH difference in the acidophilic bacteria Thermoplasma acidophilum and Bacillus acidocaldarius.

The mechanism by which acidophilic bacteria generate and maintain their cytoplasmic pH close to neutrality was investigated. For this purpose we determined the components of proton motive force in the eubacterium Bacillus acidocaldarius and the archaebacterium Thermoplasma acidophilum. After correction for probe binding, the proton motive force of untreated cells was 190 to 240 mV between external pH 2 and 4. Anoxia diminished total proton motive force and the transmembrane pH difference by 60 to 80 mV. The protonophore 2,4-dinitrophenol abolished the total proton motive force almost completely and diminished the transmembrane pH difference by at least two units. However, even after correction for probe binding, protonophore-treated cells maintained a pH difference of approximately one unit.     

Oxygen taxis and proton motive force in Azospirillum brasilense.

The microaerophilic nitrogen-fixing bacterium Azospirillum brasilense formed a sharply defined band in a spatial gradient of oxygen. As a result of aerotaxis, the bacteria were attracted to a specific low concentration of oxygen (3 to 5 microM). Bacteria swimming away from the aerotactic band were repelled by the higher or lower concentration of oxygen that they encountered and returned to the band. This behavior was confirmed by using temporal gradients of oxygen. The cellular energy level in A. brasilense, monitored by measuring the proton motive force, was maximal at 3 to 5 microM oxygen. The proton motive force was lower at oxygen concentrations that were higher or lower than the preferred oxygen concentration. Bacteria swimming toward the aerotactic band would experience an increase in the proton motive force, and bacteria swimming away from the band would experience a decrease in the proton motive force. It is proposed that the change in the proton motive force is the signal that regulates positive and negative aerotaxis. The preferred oxygen concentration for aerotaxis was similar to the preferred oxygen concentration for nitrogen fixation. Aerotaxis is an important adaptive behavioral response that can guide these free-living diazotrophs to the optimal niche for nitrogen fixation in the rhizosphere.     

The interaction between electron transfer,proton motive force and solute transport in bacteria

The properties of proton solute symport have been studied inStreptococcus cremoris, Rhodopseudomonas sphaeroides andEscherichia coli. In the homolactic fermentative organismS. cremoris the efflux of lactate is a membrane proteinmediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents inE. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. InRps. sphaeroides andE. coli the activity of several transport proteins depends on the activity of the electron transfer systems. On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.     

Relationship between proton motive force and motility in Spirochaeta aurantia.

The effects of various metabolic inhibitors on the motility of Spirochaeta aurantia were investigated. After 15 min in sodium arsenate buffer, 90% of cells remained motile even though adenosine triphosphate levels dropped from 5.6 to 0.1 nmol/mg (dry weight) of cells. After 70 min in sodium arsenate, 5% of cells were motile. Addition of phenazine methosulfate plus ascorbate at this time resulted in motility of 95% of cells, but adenosine triphosphate levels remained at 0.1 nmol/mg of cell dry weight. Carbonyl cyanide-m-chlorophenyl hydrazone rapidly (within 1 min) and completely inhibited motility of metabolizing cells in potassium phosphate buffer. However, after 15 min in the presence of carbonyl cyanide m-chlorophenyl hydrazone the cellular adenosine triphosphate level was 3.4 nmol/mg (dry weight) of cells, and the rate of oxygen uptake was 44% of the rate measured in the absence of carbonyl cyanide m-chlorophenyl hydrazone. Cells remained motile under conditions where either the electrical potential or the pH gradient across the membrane of S. aurantia was dissipated. However, if both gradients were simultaneously dissipated, motility was rapidly inhibited. This study indicates that a proton motive force, in the form of either a transmembrane electrical potential or a transmembrane pH gradient, is required for motility in S. aurantia. Adenosine triphosphate does not appear to directly activate the motility system in this spirochete.     

Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force.

The magnitude of the proton motive force generated during in vitro substrate oxidation by Coxiella burnetii was examined. The intracellular pH of C. burnetii varied from about 5.1 to 6.95 in resting cells over an extracellular pH range of 2 to 7. Similarly, delta psi varied from about 15 mV to -58 mV over approximately the same range of extracellular pH. Both components of the proton motive force increased during substrate oxidation, resulting in an increase in proton motive force from about -92 mV in resting cells to -153 mV in cells metabolizing glutamate at pH 4.2. The respiration-dependent increase in proton motive force was blocked by respiratory inhibitors, but the delta pH was not abolished even by the addition of proton ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone or 2,4-dinitrophenol. Because of this apparently passive component of delta pH maintenance, the largest proton motive force was obtained at an extracellular pH too low to permit respiration. C. burnetii appears, therefore, to behave in many respects like other acidophilic bacteria. Such responses are proposed to contribute to the extreme resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome of eucaryotic cells in which this organism multiplies.     

Quantitative measurements of proton motive force and motility in Bacillus subtilis.

The protein motive force of metabolizing Bacillus subtilis cells was only slightly affected by changes in the external pH between 5 and 8, although the electrical component and the chemical component of the proton motive force contributed differently at different external pH. The electrical component of the proton motive force was very small at pH 5, and the chemical component was almost negligible at pH 7.5. At external pH values between 6 and 7.7, swimming speed of the cells stayed constant. Thus, either the electrical component or the chemical component of the proton motive force could drive the flagellar motor. When the proton motive force of valinomycin-treated cells was quantitatively decreased by increasing the external K+ concentration, the swimming speed of the cells changed in a unique way: the swimming speed was not affected until about--100 mV, then decreased linearly with further decrease in the proton motive force, and was almost zero at about--30 mV. The rotation rate of a flagellum, measured by a tethered cell, showed essentially the same characteristics. Thus, there are a threshold proton motive force and a saturating proton motive force for the rotation of the B. subtilis flagellar motor.     

Effects of potassium ions on proton motive force in Rhodobacter sphaeroides.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (delta psi) only. At pH 7.9 the PMF was composed of a high delta psi of -98 mV and was partially compensated by a reversed pH gradient (delta pH) of +37 mV. ATPase inhibitors did not affect the delta psi, which was most likely the result of a K+ diffusion potential. Under energized conditions in the presence of K+ the delta psi depolarized due to electrogenic K+ uptake. This led to the generation of a delta pH (inside alkaline) in the external pH range of 6 to 8. This delta pH was dependent on the K+ concentration and was maximal at external K+ concentrations larger than 1.2 mM. In energized cells in 50 mM KPi buffer containing 5 mM MgSO4, a delta pH (inside alkaline) was present at external pHs from pH 6 to 8. As a result the overall magnitude of the PMF at various external pHs remained constant at -130 mV, which was significantly higher than the PMF under anaerobic-dark conditions. In the absence of K+, in 50 mM NaPi buffer containing 5 mM MgSO4, no depolarization of the delta psi was found and the PMF was composed of a large delta psi and a small delta pH. The delta pH became even reversed (inside acidic) at alkaline pHs (pH>7.3), resulting in a lowering of the PMF. These results demonstrate that in R. sphaeroides K+ uptake is essential for the generation of a delta pH and plays a central role in the regulation of the internal pH.     

Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force.

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.     

Depletion of proton motive force by nisin in Listeria monocytogenes cells.

The basal proton motive force (PMF) levels and the influence of the bacteriocin nisin on the PMF were determined in Listeria monocytogenes Scott A. In the absence of nisin, the interconversion of the pH gradient (Z delta pH) and the membrane potential (delta psi) led to the maintenance of a fairly constant PMF at -160 mV over the external pH range 5.5 to 7.0. The addition of nisin at concentrations of greater than or equal to 5 micrograms/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 microgram of nisin per ml, delta pH was completely dissipated but delta psi decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only delta pH, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only delta psi, under conditions in which nisin depleted both. Four other L. monocytogenes strains had basal PMF parameters similar to those of strain Scott A. Nisin (2.5 micrograms/ml) also completely dissipated PMF in these strains.     

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.     

Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulolytic ruminal bacterium.

The utilization of cellulose or cellobiose by Bacteroides succinogenes S85 was severely inhibited at pH values of less than 5.7. Since low pH inhibited the utilization of both cellobiose and cellulose, changes in cellulase activity could not explain the effect. At an extracellular pH of 6.9, the pH gradient (delta pH) across the cell membrane was only 0.07 U. As extracellular pH declined from 6.9 to 5.7, intracellular pH decreased to a smaller extent than extracellular pH and delta pH increased. Below pH 5.7, there was a linear and nearly proportional decrease in intracellular pH. B. succinogenes took up the lipophilic cation tetraphenylphosphonium ion (TPP+) in the presence of cellobiose, and uptake was sensitive to the ionophore valinomycin. As pH was decreased with phosphoric acid, the cells lost TPP+ and electrical potential, delta psi, decreased. From extracellular pH 6.9 to 5.7, the decrease in delta psi was compensated for by an increase in delta pH, and the proton motive force ranged from 152 to 158 mV. At a pH of less than 5.7, there was a large decrease in proton motive force, and this decrease corresponded to the inhibition of cellobiose utilization.     

Hydrogenosomes: eukaryotic adaptations to anaerobic environments.

Like mitochondria, hydrogenosomes compartmentalize crucial steps of eukaryotic energy metabolism; however, this compartmentalization differs substantially between mitochondriate aerobes and hydrogenosome-containing anaerobes. Because hydrogenosomes have arisen independently in different lineages of eukaryotic microorganisms, comparative analysis of the various types of hydrogenosomes can provide insights into the functional and evolutionary aspects of compartmentalized energy metabolism in unicellular eukaryotes.     

Effects of K+ and Na+ on the proton motive force of respiring Escherichia coli at alkaline pH.

The role of K+ and Na+ in the maintenance of the proton motive force (delta p) was studied in Escherichia coli incubated in alkaline media. Cells respiring in Tris buffer (pH 7.8) that contained less than 100 microEq of K+ and Na+ per liter had a normal delta p of about -165 mV. At pH 8.2, however, the delta p was reduced significantly. The decrease in delta p at pH 8.2 was due to a marked decrease in the transmembrane potential (delta psi), while the internal pH remained at 7.5 to 7.7. When KCl or NaCl, but not LiCl or choline chloride, was added to the cells, the delta psi rose to the values seen at an external pH of 7.8. In addition, choline chloride inhibited the enhancement of delta psi by K+. None of the salts had a significant effect on the internal pH. The effects can be attributed to alterations of K+ or Na+ cycling in and out of the cells via the known K+ and Na+ transport systems.     

Role of proton motive force in genetic transformation of Bacillus subtilis

This study explored the role of the proton motive force in the processes of DNA binding and DNA transport of genetic transformation of Bacillus subtilis 168 strain 8G-5 (trpC2). Transformation was severely inhibited by the ionophores valinomycin, nigericin, and 3,5-di-tert-4-hydroxybenzylidenemalononitrite (SF-6847) and by tetraphenylphosphonium. The ionophores valinomycin and nigericin also severely inhibited binding of transforming DNA to the cell envelope, whereas SF-6847 and carbonylcyanide-p-trifluoromethoxyphenylhydrazone hardly affected binding. The proton motive force, therefore, does not contribute to the process of DNA binding, and valinomycin and nigericin interact directly with the DNA binding sites at the cell envelope. The effects of ionophores, weak acids, and tetraphenylphosphonium on the components of the proton motive force and on the entry of transforming DNA after binding to the cell envelope was investigated. DNA entry, as measured by the amount of DNase I-resistant cell-associated [3H]DNA and by the formation of DNA breakdown products, was severely inhibited under conditions of a small proton motive force and also under conditions of a small delta pH and a high electrical potential. These results suggest that the proton motive force and especially the delta pH component functions as a driving force for DNA uptake in transformation.     

Role of proton motive force in phototactic and aerotactic responses of Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides grown under nonrigorous anaerobic conditions in the light developed components of a branched respiratory electron transfer chain, and a photosynthetic electron transfer chain. Both respiratory pathways were sensitive to rotenone and high concentrations of cyanide, but oxygen uptake was only partially inhibited by the addition of low concentrations of cyanide or antimycin A. When incubated anaerobically in the dark, R. sphaeroides responded positively to an oxygen gradient in the absence of rotenone. In the presence of rotenone, aerotaxis only occurred when the antimycin A-sensitive branch of the pathway was functioning, although both branches still reduced oxygen. Although there was electron movement along the respiratory chain, aerotaxis only occurred in response to a change in proton motive force. When incubated anaerobically in the light, the movement of R. sphaeroides up a light gradient depended on photosynthetic electron transport. When incubated aerobically, high-intensity actinic illumination inhibited oxygen uptake and aerotaxis. In a low-intensity light gradient the phototactic response was inhibited by oxygen. These results are discussed in relation to the interaction of the electron transfer chains and their roles in controlling tactic responses in R. sphaeroides.     

In vivo regulation of thylakoid proton motive force in immature leaves

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.