Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

Evidence for a chemiosmotic mechanism of ATP synthesis in methanogenic bacteria

Recent studies on methanogenesis from methanol and H₂ in Methanosarcina barkeri have provided the first proof of a chemiosmotic mechanism of ATP synthesis in methanogenic bacteria.     

Discrimination between transmembrane ion gradient-driven and electron transfer-driven ATP synthesis in the methanogenic bacteria

As with Methanococcus voltae [(1986) FEBS Lett. 200, 177–180], ATP synthesis in Methanobacterium thermoautotrophicum (ΔH) can be driven by the imposition of a sodium gradient, but only in the presence of a counterion. Monensin (but not SF6847) inhibits this synthesis. Methanogenic electron transfer-driven ATP synthesis, however, is insensitive to the combination of these two ionophores. In M. voltae, 117 μM diethylstilbestrol effectively inhibits both membrane potential- and sodium gradient-driven ATP synthesis, but has no effect on ATP production coupled to methanogenesis. In Mb. thermoautotrophicum (ΔH), a similar pattern of inhibition is exhibited by harmaline, an inhibitor of sodium-linked membrane transport systems. We conclude that ATP-driven sodium translocation and electron transfer-driven ATP synthesis are accomplished by separate entities, at least for these two only distantly related species of methanogen.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

A tridecameric c ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential

Despite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton-coupled F(1)F(o)-ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c-subunits using a novel mass spectrometry method, termed 'laser-induced liquid bead ion desorption' (LILBID). This technique allows the mass determination of non-covalently assembled, detergent-solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential.     

Ammonium/urea-dependent generation of a proton electrochemical potential and synthesis of ATP in Bacillus pasteurii.

The influence of ammonium and urea on the components of the proton electrochemical potential (delta p) and de novo synthesis of ATP was studied with Bacillus pasteurii ATCC 11859. In washed cells grown at high urea concentrations, a delta p of -56 +/- 29 mV, consisting of a membrane potential (delta psi) of -228 +/- 19 mV and of a transmembrane pH gradient (delta pH) equivalent to 172 +/- 38 mV, was measured. These cells contained only low amounts of potassium, and the addition of ammonium caused an immediate net decrease of both delta psi and delta pH, resulting in a net increase of delta p of about 49 mV and de novo synthesis of ATP. Addition of urea and its subsequent hydrolysis to ammonium by the cytosolic urease also caused an increase of delta p and ATP synthesis; a net initial increase of delta psi, accompanied by a slower decrease of delta pH in this case, was observed. Cells grown at low concentrations of urea contained high amounts of potassium and maintained a delta p of -113 +/- 26 mV, with a delta psi of -228 +/- 22 mV and a delta pH equivalent to 115 +/- 20 mV. Addition of ammonium to such cells resulted in the net decrease of delta psi and delta pH without a net increase in delta p or synthesis of ATP, whereas urea caused an increase of delta p and de novo synthesis of ATP, mainly because of a net increase of delta psi. The data reported in this work suggest that the ATP-generating system is coupled to urea hydrolysis via both an alkalinization of the cytoplasm by the ammonium generated in the urease reaction and a net increase of delta psi that is probably due to an efflux of ammonium ions. Furthermore, the findings of this study show that potassium ions are involved in the regulation of the intracellular pH and that ammonium ions may functionally replace potassium to a certain extent in reducing the membrane potential and alkalinizing the cytoplasm.     

ATP synthesis in Staphylococcus aureus cells during induction of membrane potentials and proton gradient

The dynamics of ATP synthesis in Staphylococcus aureus cells was studied during membrane potential induction and K+ gradient generation in the presence of valinomycin. The starting level of intracellular ATP was 0.05 mM. Valinomycin (30 micrograms/ml) caused an increment of the intracellular ATP level up to 0.25 mM. The protonophore uncoupler, m-chlorinecarbonylcyanidephenylhydrazonium, and the H+-ATPase inhibitor, N,N'-dicyclohexylcarbodiimide, effectively suppress ATP synthesis induced by valinomycin. No ATP synthesis occurs at K+ concentration of 200 mM. The transmembrane gradient formation results in the synthesis of a smaller amount of ATP (0.10 mM).     

Millisecond kinetics of ATP synthesis driven by externally imposed electrochemical potentials in chloroplasts

We have used rapid mixing and quenching techniques to measure the initial ATP synthesis rates and the duration of the ATP synthetic capacity derived from artificially imposed proton gradients and valinomycin-mediated K+ diffusion potentials in chloroplasts. The initial rate of ATP synthesis driven by a K+ diffusion potential was 10-fold slower than that driven by an acid-base transition of equivalent electrochemical potential. Total yields of ATP resulting from a K+ concentration shift were only slightly affected by the absence of Cl-, indicating that Cl- permeability does not significantly reduce the K+ diffusion potential. The ATP synthetic capacity decayed with a half-life of 0.2 s in the case of a K+ diffusion potential and a half-life of 1.0 s in the case of an acid-base shift. In both cases, ATP, added at the time of the pH or [KCl] shift, slowed the decay of the ATP synthesis rates, indicating that the coupling factor controls a channel for proton efflux, as proposed earlier (Portis, A.R., and McCarty, R.E. (1974) J. Biol. Chem. 249, 6250-6254). Because the proton gradient has a longer half-life than the K+ diffusion potential, when combinations of the two are employed to drive ATP synthesis, the proton gradient will make a larger contribution to the initial rate and total yield than that predicted from a strictly linear proportionality of the initial magnitudes of the two gradients.     

Studies on the relationship between ATP synthesis and transport and the proton electrochemical gradient in rat liver mitochondria

The effect of ATP synthesis on delta mu H in rat liver mitochondria has been analyzed by separating the steps of adenine nucleotide translocation and ATP synthesis in the matrix. Either exchange of ATP, synthesized by substrate level phosphorylation in the matrix of oligomycin-treated mitochondria, for external ADP, or activity of the membrane-bound ATP synthase complex results in delta mu H depression with respect to resting state levels. This depression appears to be more pronounced, under strictly comparable conditions, when arsenate is used to stimulate ATP synthase activity than when the ornithine-citrulline conversion reaction is used for the same purpose.     

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.     

An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.     

Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser

Particulate adenylate cyclase (AC) and guanylate cyclase (GC) activities localized in the ciliary membrane from Paramecium were solubilized by a two-step procedure using the detergents Brij 56 and Lubrol PX. The enzymes remained in the supernatant after a 100 000 × g centrifugation. Upon gel chromatography, AC and GC were almost completely separated proving that each enzyme is a distinct molecular entity. Solubilization of GC was achieved with the calmodulin subunit remaining firmly attached to the catalytic part. Antibodies against calmodulin inhibited the enzyme as did La³⁺ and EGTA. AC activity appeared to be regulated specifically by K⁺, enzyme activity being enhanced up to 100% by 15 mM K⁺. Na⁺ and Li⁺ were inactive.     

Quantification of the electrochemical proton gradient and activation of ATP synthase in leaves

We have developed a new method to quantify the transmembrane electrochemical proton gradient present in chloroplasts of dark-adapted leaves. When a leaf is illuminated by a short pulse of intense light, we observed that the light-induced membrane potential changes, measured by the difference of absorption (520 nm-546 nm), reach a maximum value (approximately 190 mV) determined by ion leaks that occur above a threshold level of the electrochemical proton gradient. After the light-pulse, the decay of the membrane potential follows a multiphasic kinetics. A marked slowdown of the rate of membrane potential decay occurs approximately 100 ms after the light-pulse, which has been previously interpreted as reflecting the switch from an activated to an inactivated state of the ATP synthase (Junge, W., Rumberg, B. and Schr?der, H., Eur. J. Biochem. 14 (1970) 575-581). This transition occurs at approximately 110 mV, thereby providing a second reference level. On this basis, we have estimated the Delta micro (H(+)) level that pre-exists in the dark. Depending upon the physiological state of the leaf, this level varies from 40 to 70 mV. In the dark, the Delta micro (H(+)) collapses upon addition of inhibitors of the respiratory chain, thus showing that it results from the hydrolysis of ATP of mitochondrial origin. Illumination of the leaf for a period longer than several seconds induces a long-lived Delta micro (H(+)) increase (up to approximately 150 mV) that reflects the light-induced increase in ATP concentration. Following the illumination, Delta micro (H(+)) relaxes to its dark-adapted value according a multiphasic kinetics that is completed in more than 1 h. In mature leaf, the deactivation of the Benson-Calvin cycle follows similar kinetics as Delta micro (H(+)) decay, showing that its state of activation is mainly controlled by ATP concentration.     

An explanation of apparent sudden change in the activation energy of membrane enzymes.

If an enzyme-lipid mixture forms phases of pure lipid and enzyme-lipid solution, and enzyme activity depends on the composition of the enzyme-lipid solution, the temperature-dependence of lipid solubility in the enzyme-lipid solution leads to apparent sudden changes in enzyme activation energy without activity discontinuities at lipid phase transition temperatures.     

ATP synthesis by ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716 by ionophore-induced electric potentials and proton gradients

ATP synthesis driven by low pre-established electric potentials and pH gradients is studied in large ATPase proteoliposomes, prepared from the ATPase complex and native lipids from the thermophilic cyanobacterium Synechococcus 6716. Electric potentials and pH gradients were achieved by valinomycin and nigericin, respectively, in the presence of a K⁺ gradient across the membrane. External base-pulses were also applied. In this system ATP synthesis driven by valinomycin-induced K⁺ influx, nigericin-induced internal acidification and by external base-pulses is demonstrated. Electric potentials and pH gradients of equivalent size lead to roughly similar ATP synthesis activities. ATP synthesis is optimal at 80–100 nM valinomycin and at 0.75−1 μM nigericin at the proper pre-set ion gradients. Uncoupler and DCCD inhibit ATP synthesis. Prior activation of the complex by thiol agents or trypsin was not required for synthesis activity. The ATP synthesis rate increases with the size of electric potential or pH gradient. The threshold value of the electrochemical gradient for significant ATP synthesis is about 30 mV. ATP production proceeds for more than 60 min. The generation of ionophore-induced electric potentials and pH gradients have been followed by oxonol VI and intraliposomal Neutral red, respectively. The extent of the absorbance changes of both probes is proportional to the size of electric potential or pH gradient. Ionophore-induced oxonol VI and Neutral red responses are stable for at least 30 min. The results are discussed in terms of membrane permeability and vesicle size.     

An analysis of proton fluxes coupled to electron transport and ATP synthesis in chloroplast thylakoids

Electron transport, phosphorylation and internal proton concentration were measured in illuminated spinach chloroplast thylakoid membranes under a number of conditions. Regardless of the procedure used to vary these parameters, the data fit a simple chemiosmotic model. Protons from Photosystem II did not appear to be utilized differently from those derived from Photosystem I. The maximal phosphorylation efficiency ($\text{P}\text{e}{}_2$) for photophosphorylation in washed thylakoids under oxidizing conditions is likely to be $\text{4}\text{3}$. This value is consistent with a proton-to-electron-pair ratio of 4 for electron flow through both photosystems and a proton-to-ATP ratio of 3 for the chloroplast proton-ATPase.