Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.     

The sodium cycle in methanogenesis. CO2 reduction to the formaldehyde level in methanogenic bacteria is driven by a primary electrochemical potential of Na+ generated by formaldehyde reduction to CH4

CH4 formation from CO2 and H2 rather than from formaldehyde and H2 in methanogenic bacteria is inhibited by uncouplers, indicating that CO2 reduction to the formaldehyde level is energy-driven. We report here that in Methanosarcina barkeri the driving force is a primary electrochemical sodium potential (delta mu Na+) generated by formaldehyde reduction to CH4. This is concluded from the following findings. 1. CO2 reduction to CH4 was insensitive towards protonophores, when the Na+/H+ antiporter was inhibited; under these conditions delta mu Na+ was 120 mV (inside negative), whereas both delta mu H+ and the cellular ATP content were low. 2. CO2 reduction to CH4, rather than formaldehyde reduction, was sensitive towards Na+ ionophores, which dissipated delta mu Na+. 3. CO2 reduction to CH4, in the presence of protonophores and Na+/H+ antiport inhibitors, was coupled with the extrusion of 1-2 mol Na+/mol CH4, and formaldehyde reduction to CH4 was coupled with the extrusion of 3-4 mol Na+/mol CH4. Thus during CO2 reduction to the formaldehyde level 2-3 mol Na+ were consumed.     

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.     

ATP synthesis in Methanobacterium thermoautotrophicum coupled to CH4 formation from H2 and CO2 in the apparent absence of an electrochemical proton potential across the cytoplasmic membrane

Methanogenic bacteria are considered to couple methane formation with the synthesis of ATP by a chemiosmotic mechanism. This hypothesis was tested with Methanobacterium thermoautotrophicum. Methane formation from H2 and CO2 (2.5 - 3 mumol X min-1 X mg cells-1) by cell suspensions of this organism resulted in the formation of an electrochemical proton potential (delta mu H +) across the cytoplasmic membrane of 230 mV (inside negative) and in the synthesis of ATP up to an intracellular concentration of 5 - 7 nmol/mg. The addition of ionophores at concentrations which completely dissipated delta mu H + without inhibiting methane formation did not result in an inhibition of ATP synthesis. It thus appears that delta mu H + across the cytoplasmic membrane is not the driving force for the synthesis of ATP in M. thermoautotrophicum.     

Mechanisms of sodium transport in bacteria

In some bacteria, an Na+ circuit is an important link between exergonic and endergonic membrane reactions. The physiological importance of Na+ ion cycling is described in detail for three different bacteria. Klebsiella pneumoniae fermenting citrate pumps Na+ outwards by oxaloacetate decarboxylase and uses the Na+ ion gradient thus established for citrate uptake. Another possible function of the Na+ gradient may be to drive the endergonic reduction of NAD+ with ubiquinol as electron donor. In Vibrio alginolyticus, an Na+ gradient is established by the NADH: ubiquinone oxidoreductase segment of the respiratory chain; the Na+ gradient drives solute uptake, flagellar motion and possibly ATP synthesis. In Propionigenium modestum, ATP biosynthesis is entirely dependent on the Na+ ion gradient established upon decarboxylation of methylmalonyl-CoA. The three Na(+)-translocating enzymes, oxaloacetate decarboxylase of Klebsiella pneumoniae, NADH: ubiquinone oxidoreductase of Vibrio alginolyticus and ATPase (F1F0) of Propionigenium modestum have been isolated and studied with respect to structure and function. Oxaloacetate decarboxylase consists of a peripheral subunit (alpha), that catalyses the carboxyltransfer from oxaloacetate to enzyme-bound biotin. The subunits beta and gamma are firmly embedded in the membrane and catalyse the decarboxylation of the carboxybiotin enzyme, coupled to Na+ transport. A two-step mechanism has also been demonstrated for the respiratory Na+ pump. Semiquinone radicals are first formed with the electrons from NADH; subsequently, these radicals dismutate in an Na(+)-dependent reaction to quinone and quinol. The ATPase of P. modestum is closely related in its structure to the F1F0 ATPase of E. coli, but uses Na+ as the coupling ion. A specific role of protons in the ATP synthesis mechanism is therefore excluded.     

Sodium ion-translocating decarboxylases

The review is concerned with three Na(+)-dependent biotin-containing decarboxylases, which catalyse the substitution of CO(2) by H(+) with retention of configuration (DeltaG degrees '=-30 kJ/mol): oxaloacetate decarboxylase from enterobacteria, methylmalonyl-CoA decarboxylase from Veillonella parvula and Propiogenium modestum, and glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. The enzymes represent complexes of four functional domains or subunits, a carboxytransferase, a mobile alanine- and proline-rich biotin carrier, a 9-11 membrane-spanning helix-containing Na(+)-dependent carboxybiotin decarboxylase and a membrane anchor. In the first catalytic step the carboxyl group of the substrate is converted to a kinetically activated carboxylate in N-carboxybiotin. After swing-over to the decarboxylase, an electrochemical Na(+) gradient is generated; the free energy of the decarboxylation is used to translocate 1-2 Na(+) from the inside to the outside, whereas the proton comes from the outside. At high [Na(+)], however, the decarboxylases appear to catalyse a mere Na(+)/Na(+) exchange. This finding has implications for the life of P. modestum in sea water, which relies on the synthesis of ATP via Delta(mu)Na(+) generated by decarboxylation. In many sequenced genomes from Bacteria and Archaea homologues of the carboxybiotin decarboxylase from A. fermentans with up to 80% sequence identity have been detected.     

Na(+)-coupled alternative to H(+)-coupled primary transport systems in bacteria.

Protons are the most common coupling ions in bacterial energy conversions. However, while many organisms, such as the alkaliphilic Bacilli, employ H(+)-bioenergetics for electron transport phosphorylation, they use Na+ as the coupling ion for transport and flagellar movement. The Na+ gradient required for these bioenergetic functions is established by the secondary Na+/H+ antiporter. In contrast, Vibrio alginolyticus and methanogenic bacteria have primary pumps for both H+ and Na+. They use the proton gradient for ATP synthesis while other, less energy-consuming membrane reactions are powered by the Na+ gradient. In a third mode, some anaerobic bacteria possess decarboxylases acting as primary Na+ pumps. For instance, in Klebsiella pneumoniae, the Na+ gradient established by oxaloacetate decarboxylase is used for the uptake of the growth substrate citrate, and Propionigenium modestum consumes the energy of the Na+ gradient formed by methylmalonyl-CoA decarboxylase directly for ATP synthesis.     

On the mechanism of sodium ion translocation by methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7-8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl-CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating delta psi of 60 mV, delta pNa+ of 50 mV, and delta mu Na+ of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions malonyl-CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl-CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl-CoA decarboxylation rate 2.8-fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8-fold. Methylmalonyl-CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl-CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl-CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.     

Chemiosmotic systems in bioenergetics: H(+)-cycles and Na(+)-cycles.

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H+ cycle concept. The circulation of H+ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H+ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes. Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H+. It was found that, in certain bacteria, as well as in the outer membrane of the animal cell, Na+ effectively substitutes for H+ as the coupling ion (the chemiosmotic Na+ cycle). A precedent is set when the Na+ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H+ and Na+ cycles coexist in one and the same membrane (bacteria) or in two different membranes of one and the same cell (animals). The sets of delta mu H+ and delta mu Na+ generators as well as delta mu H+ and delta mu Na+ consumers found in different types of biomembranes, are listed and discussed.     

Free energy coupling between H+-generating and H+-consuming pumps. Ratio between output and input forces

The delta Gp/delta mu H ratio has been measured in mitochondria close to state 4 in the presence of various uncoupler or K+/valinomycin concentrations in media containing either 1 mM or 50 mM Pi. Care has been taken to control the factors affecting delta Gp and delta mu H which could lead to an artefactual increase of the delta Gp/delta mu H ratio above the highest accepted value for the H+/ATP stoichiometry (n = 4, synthesis + transport). In particular, to avoid overestimation of delta Gp due to inactivation of the ATPases at low delta mu H or to the presence of adenylate kinase, the static head state was approached from the side of net ATP synthesis and delta Gp was measured in a state close to static head but still maintaining a residual rate of aerobic phosphorylation. For each concentration of uncoupler or K+, the Pi concentration and/or the adenylate energy charge (EC) as a function of time have been measured as indicators of net ATP synthesis. Only the values of delta Gp measured during a decrease in Pi concentration and/or an increase in EC have been considered to be meaningful for calculations of delta Gp/delta mu H ratios. Both uncouplers and K+ transport cause a marked depression of delta mu H and a parallel depression of the rate of ATP synthesis. However the low rate of ATP synthesis taking place under conditions of low delta mu H eventually results, especially at high Pi concentrations, in a relatively large delta Gp. The delta Gp/delta mu H ratios obtained at the lower delta mu H values exceed 4 and approach 6. Although slightly higher delta Gp/delta mu H ratios are obtained with valinomycin-treated than with uncoupler-treated mitochondria, the pattern of the rise of the force ratio as delta mu H decreases is similar in both cases. An increase of the delta Gp/delta mu H ratio above 4, the maximal accepted H+/ATP stoichiometry is thermodynamically incompatible with the delocalized protonic coupling model.     

Sodium-coupled ATP synthesis in the bacterium Vitreoscilla.

The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla.     

The membrane potential ofMethanobacterium thermoautotrophicum under different external conditions

The membrane potential (delta psi) of whole cells of Methanobacterium thermoautotrophicum strain delta H was estimated under different external conditions using a TPP(+)-sensitive electrode. The results show that the delta psi values of M. thermoautotrophicum at alkaline pHout (8.5) are comparable with delta psi values under slightly acidic conditions (pH 6.8; 230 and 205 mV, respectively). On the other hand, the size of colonies on Petri dishes was remarkably smaller at pH 8.5 than at 6.8. The delta psi was insensitive to relevant ATPase inhibitors. At pH 6.8, the protonophore 3,3',4',5-tetrachlorosalicylanilide (TCS) strongly inhibited delta psi formation and ATP synthesis driven by methanogenic electron transport. On the other hand, at pH 8.5 the CH4 formation and ATP synthesis were insensitive to TCS and a protonophore-resistant delta psi of approximately 150 mV was determined. The finding of a protonophore-resistant delta psi at pH 8.5 indicates that at alkaline pHout these cells can switch from H(+)-energetics to Na(+)-energetics, when the delta [symbol: see text] H+ becomes limited. The results strongly support the hypothesis that at alkaline pHout Na+ ions might fully substitute for H+ in these cells as the coupling ions.     

Voltage-generated torque drives the motor of the ATP synthase.

The mechanism by which ion-flux through the membrane-bound motor module (F0) induces rotational torque, driving the rotation of the gamma subunit, was probed with a Na+-translocating hybrid ATP synthase. The ATP-dependent occlusion of 1 (22)Na+ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22Na+ was added first and ATP second, but not if the order of addition was reversed. These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22Na+-loaded or a DCCD-modified rotor subunit reaches the Na+-impermeable stator. The ATP synthase with a Na+-permeable stator catalyzed 22Na+out/Na+in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (DeltaPsi) of 90 mV. We propose that in the idling mode of the motor, Na+ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator. This motional flexibility is arrested if either ATP or DeltaPsi induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with DeltaPsi of 60-125 mV but not with DeltapNa+ of 195 mV. These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor.     

The sodium ion translocating adenosinetriphosphatase of Propionigenium modestum pumps protons at low sodium ion concentrations

The purified ATPase (F1F0) of Propionigenium modestum has its pH optimum at pH 7.0 or at pH 6.0 in the presence or absence of 5 mM NaCl, respectively. The activation by 5 mM NaCl was 12-fold at pH 7.0, 3.5-fold at pH 6.0, and 1.5-fold at pH 5.0. In addition to its function as a primary Na+ pump, the ATPase was capable of pumping protons. This activity was demonstrated with reconstituted proteoliposomes by the ATP-dependent quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine. No delta pH was formed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or by blocking the ATPase with dicyclohexylcarbodiimide. In the presence of valinomycin and K+, the delta pH increased, in accord with the operation of an electrogenic proton pump. The proton pump was only operative at low Na+ concentrations (less than 1 mM), and its activity increased as the Na+ concentration decreased. Parallel to the decrease of H+ pumping, the velocity of the Na+ transport increased about 6-fold from 0.1 to 4 mM NaCl, indicating a switch from H+ to Na+ pumping, as the Na+ concentration increases. Due to proton leaks in the proteoliposomal membranes, fluorescence quenching was released after blocking the ATPase with dicyclohexylcarbodiimide, by trapping residual ATP with glucose and hexokinase, or by the Na+-induced conversion of the proton pump onto a Na+ pump. Amiloride, an inhibitor of various Na+-coupled transport systems, was without effect on the kinetics of Na+ transport by the P. modestum ATPase.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

The sodium cycle--a new type of bacterial energetics

The literature data and experimental results of the author's laboratory on the role of Na+ in bacterial energetics are reviewed. It was shown that certain bacterial species utilize the transmembrane difference of Na+ electrochemical potentials (delta mu Na+) as a convertible membrane-linked form of energy. The membranes of such bacteria were found to contain delta mu Na+ generators (e. g., decarboxylases of some carboxylic acids of NADH-menaquinone reductase). It was shown that delta mu Na+ formed by these generators may support all the three main types of work of the bacterial cell, i. e., chemical (ATP synthesis), osmotic (substrate accumulation) and mechanical (motility).     

The role of sodium ions in methanogenesis. Formaldehyde oxidation to CO2 and 2H2 in methanogenic bacteria is coupled with primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (delta G0' = -27 kJ/mol CO2), when methanogenesis was inhibited by 2-bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings. Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2-3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores of Na+/H+ antiport inhibitors. Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores. Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide. With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.     

Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4.

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.