Miloslav Greksák

The presence of H+ and Na+‐translocating ATPases in Methanobacterium thermoautotrophicum and their possible function under alkaline conditions

Two ATPases with different apparent molecular masses of approx. 500 kDa and 400 kDa were identified in the EDTA extract of the cell membranes of Methanobacterium thermoautotrophicum. Western blotting with polyclonal antiserum reactive with β‐subunit of mitochondrial ATPase from rat liver and yeast was used for further analysis of these ATPases. A strong crossreactivity with a single protein band with an apparent molecular weight of about 53 kDa (similar to β‐subunit of F‐type ATPase from other sources) was found in protein extracts of whole cells of Methanobacterium thermoautotrophicum strains ΔH and Marburg, as well as of Methanospirillum hungatei. This indicates the presence of F‐type ATPase in methanogens. ATP synthesis driven by membrane potential which was generated by artificially‐imposed ΔpH in the presence of protonophorous uncoupler and sodium ions was stimulated by bafilomycin A1, an inhibitor of V‐ and A‐type ATPases, as well as by harmaline, an inhibitor of Na+/H+ antiporter. These results indicate that cells of Methanobacterium thermoautotrophicum strain ΔH contain the F‐type ATP synthase which is Na+‐translocating in addition to V‐ or A‐type ATP synthase which is H+‐translocating.

Na+-driven ATP synthesis in Methanobacterium thermoautotrophicum can be modulated with sodium ion concentrations in the growth medium

Methane formation by nongrowing cells of Methanobacterium thermoautotrophicum ΔH can be enhanced with sodium ions only when the cells were originally grown in the presence of a low (5 mM) concentration of NaCl. ATP synthesis in the cells driven by an artificially imposed Na+ gradient can be modulated by changing the sodium ion concentration in the growth medium while H+ gradient‐driven ATP synthesis does not depend on it. Stimulation of the entire ATPase activity with Na+ in permeabilized cells depends also on the Na+ concentration in the growth medium. The results obtained might indicate the presence of an inducible Na+‐translocating ATPase in Methanobacterium thermoautotrophicum.

Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G.

Rhodamine 6G (3 μM) effectively inhibited ΔpH‐driven ATP synthesis in Methanobacterium thermoautotrophicum while ΔpNa‐driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg2+‐stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis‐driven ATP synthesis at pH 6.8 of cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis‐driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium‐motive force‐driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively in alkaline conditions and it might be the sole ATP synthesizing system when the proton‐motive force‐supported ATP synthesis is inhibited by rhodamine 6G.

Effect of 2,4-dinitrophenol and ionophores on growth and methanogenesis inMethanobacterium thermoautotrophicum

Abstract2,4-Dinitrophenol and gramicidin D completely inhibited growth and methanogenesis inMethanobacterium thermoautotrophicum. At low K+ concentrations valinomycin inhibited growth and methanogenesis relatively slightly, at high K+ concentrations (0.1m KCl) growth was inhibited completely and methanogenesis by about 50%. Monensin and nigericin inhibited growth completely; methanogenesis was inhibited like with valinomycin at high K+ concentrations. The results can be interpreted in terms of Mitchell’s chemiosmotic theory as follows. The protonmotive force inM. thermoautotrophicum is the basic source of energy for endergonic processes. Dissipation of the electrical component of protonmotive force may probably be compensated by an increased generation of the proton gradient. However, the osmotic component is essential for growth ofM. thermoautotrophicum.

Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase)

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+‐translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three‐fold as compared with the wild‐type strain. In contrast to wild‐type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation rate of the mutant cells at pH 7.5 under non‐growing conditions was nearly twice that of the wild‐type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild‐type under the same conditions, and contrary to the wild‐type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild‐type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (−223 mV and −230 mV respectively). The Mg2+‐dependent ATPase activity of the 105×g supernatant of broken cells from the mutant cells was 30% higher than in the wild‐type. On the other hand, two bands with Mg2+‐dependent ATPase activity were identified by native PAGE in this fraction in both wild‐type as well as in mutant. These data suggest that the binding of Na+‐translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.

Mode of sodium ion action on methanogenesis and ATPase of the moderate halophilic methanogenis bacterium Methanohalophilus halophilus

Cells of Methanohalophilus halophilus swelled when exposed to hypotonic solutions of NaCl at pH 7.0. The swelling of the cells ceased in the presence of Mg2+. Methane formation by non‐growing cells was strongly dependent on the NaCl concentration. Among other monovalent and divalent cations only Li+ and Mg2+ could partly substitute for a specific function of sodium ions. The artificial Na+/H+ antiporter, monensin, exerted a strong inhibitory effect on methane formation from methylamine. The membrane‐bound Mg2+‐stimulated ATPase of these cells was enhanced at low (40 mM) NaCl concentration while higher concentrations of this solute were inhibitory. The results obtained show that sodium ions are a prerequisite for optimal methane formation and ATPase activity in these cells. However, both of these processes required different sodium ion concentrations.

A study of the Na /H antiport in the archaeon Methanobacterium thermoautotrophicum strain ΔH

The ability of the cells of Mb. thermoautotrophicum strain ΔH to generate a proton gradient (driven by a concentration gradient of sodium ions) at pH 6.8 as well as at pH 8 was demonstrated. The electrogenic Na+/H+ antiport responsible for this process was shown to be inhibited by EIPA and also by DCCD. Artificially increasing of intracellular concentration of Ca2+ in these cells enhanced the Na+/H+ antiport activity, while the lowering of external Ca2+ by EGTA significantly decreased this activity. The apparent Km values for Na+ about 14 and 3 mM at pH 6.8 and 8, respectively, and Vmax about 214 (pH 6.8) and 155 (pH 8) ΔQ/min per mg of cell proteins, respectively, were calculated. It is concluded that the described processes are mediated by the Na+/H+ antiporter which might be a clue to the adaptive bioenergetic behaviour of the cells of Mb. thermoautotrophicum strain ΔH under the different physiological conditions.

The presence of H+ and Na+ -linked Ca2+ extruding systems in Methanobacterium thermoautotrophicum.

The effects of monovalent cations (Na+, K+ and choline+) and the uncoupler 3,3′,4′,5‐tetrachlorosalicylanilide (TCS) were tested on 45Ca2+ uptake by non‐energized cells of Methanobacterium thermoautotrophicum. 45Ca2+ uptake was stimulated by the addition of K+ and (less) by choline+ while Na+ slowed down and even reversed it, thereby mimicking the energization of cells. The uncoupler agent, TCS, suppressed 45Ca2+ uptake in non‐energized cells in the presence or absence of Na+ but in cells energized in an atmosphere of CO2+H2 it exerted a stimulating effect. Uncoupled 45Ca2+ efflux was measured in cells pre‐loaded with 45Ca2+ by means of the divalent ionophore A23187 following its washing out by buffer containing serum albumin. The efflux was temperature‐dependent and was stimulated by external 40Ca2+ and Na+. In the absence of Na+, the uncoupled efflux was completely inhibited by TCS, whereas in the presence of Na+, TCS was without any effect. The results are in agreement with the model in which the Ca2+ influx pathway is represented by a membrane potential‐driven uniport whereas Ca2+ efflux is mediated by two transport systems ‐ and antiporters ‐ whose participation in the total efflux is dependent on the energy of the corresponding gradients of driving ions.

The membrane potential ofMethanobacterium thermoautotrophicum under different external conditions

AbstractThe membrane potential (Δψ) of whole cells ofMethanobacterium thermoautotrophicum strain ΔH was estimated under different external conditions using a TPP+-sensitive electrode. The results show that the Δψ values ofM. thermoautotrophicum at alkaline pHout (8.5) are comparable with Δψ 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 Δψ was insensitive to relevant ATPase inhibitors. At pH 6.8. the protonophore 3,3′,4′,5-tetrachlorosalicylanilide (TCS) strongly inhibited Δψ 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 Δψ of approximately 150 mV was determined. The finding of a protonophore-resistant Δψ at pH 8.5 indicates that at alkaline pHout these cells can switch from H+-energetics to Na+-energetics, when the