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.     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide     

Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum

Cells of Methanobacterium thermoautotrophicum were fixed with glutaraldehyde, sectioned and labeled with antibodies against the subunit of component C (=methyl-CoM reductase) of methyl-CoM reductase system and with colloidal gold-labeled protein A. It was found that the gold particles were located predominantly in the vicinity of the cytoplasmic membrane, when the cells were grown under conditions where methyl-CoM reductase was not overproduced. This finding confirms the recent data obtained with Methanococcus voltae showing via the same immunocytochemical localization technique that in this organism methyl-CoM reductase is membrane associated.     

Presence of a Na+/H+ antiporter in Methanobacterium thermoautotrophicum and its role in Na+ dependent methanogenesis

Methane formation from H₂/CO₂ by methanogenic bacteria is dependent on Na⁺ ions. In this communication it is shown with Methanobacterium thermoautotrophicum that a Na⁺/H⁺ antiporter plays a role in methane formation from H₂ and CO₂ and in the regulation of the ΔpH. This is based on the following findings:

1. Li⁺ ions, an alternative substrate of Na⁺/H⁺ antiporters, could replace Na⁺ in stimulating methanogenesis from H₂ and CO₂.
2. Harmaline, amiloride, and NH ₄ ⁺ , which are inhibitors of Na⁺/H⁺ antiporters, inhibited methanogenesis; inhibition was competitive to Na⁺ or Li⁺.
3. Addition of Na⁺ or Li⁺ rather than of other cations to cell suspensions resulted in an acidification of the suspension medium. The rate and extent of acidification was affected by those inhibitors, which inhibited methanogenesis competitively to Na⁺ or Li.
4. During methane formation from H₂ and CO₂ the generation of a ΔpH (inside alkaline) was dependent on the presence of Na⁺ or Li⁺. However, methanogenesis was also dependent on Na⁺ or Li⁺ under conditions where ΔpH was zero.
5. ATP synthesis driven by an electrogenic potassium efflux was significantly enhanced in the presence of Na⁺ or Li⁺. Na⁺ or Li⁺ were shown to prevent acidification of the cytoplasm under these conditions.

     

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.     

Monensin and gramicidin stimulate CH4 formation from H2 and CO2 in Methanobacterium thermoautotrophicum at low external Na+ concentration

Methane formation from H₂ and CO₂ in methanogenic bacteria is a Na⁺-dependent process. In this communication the effects of Na⁺ ionophores, of uncouplers, and of Na⁺/H⁺ antiporter inhibitors on methane formation from H₂ and CO₂ were studied with Methanobacterium thermoautotrophicum.

1. Na⁺ ionophores (the Na⁺/H⁺ antiporters monensin and lasalocid and the Na⁺ uniporter gramicidin) stimulated methanogenesis at lwo external Na⁺ concentrations when the K⁺ concentration was high. The ionophores had no effect at high external Na⁺ concentrations and were inhibitory at low external K⁺ concentrations.
2. Uncouplers (protonophores and valinomycin plus K⁺) inhibited methanogenesis at low external Na⁺ concentration at both low and high external K⁺ concentrations. Inhibition by uncouplers was relieved by the addition of either Na⁺ or Na⁺ ionophores.
3. Na⁺/H⁺ antiporter inhibitors (harmaline, amiloride, and NH ₄ ⁺ ) inhibited methanogenesis at low external Na⁺ concentration. Inhibition was relieved by the addition of either Na⁺ or of the Na⁺ ionophores.

The results are discussed with respect to the role of Na transport across the cytoplasmic membrane in methanogenesis from H₂ and CO₂.