Recording and Simulating Proton-Related Metabolism in Bacterial Cell Suspensions

Abstract

Proton release and uptake induced by metabolic activities were measured in non-buffered cell suspensions by means of a pH electrode. Recorded data were used for simulating substrate turnover rates by means of a new freeware app (proton.exe). The program applies Michaelis-Menten or first-order kinetics to the metabolic processes and allows for parametrization of simultaneously ongoing processes. The simulation includes changes of the transmembrane ΔpH, membrane potential and ATP gains, and demonstrates the principles of chemiosmotic energy conservation. In our experiments, the versatile sulfate-reducing bacterium Desulfovibrio desulfuricans CSN (DSM 9104) was used as model organism. We analysed sulfate uptake by proton-sulfate symport, scalar alkalinization by sulfate reduction to sulfide, as well as nitrate and nitrite reduction to ammonia, and electron transport-coupled proton translocation. Two types of experiments were performed: In oxidant pulse experiments, cells were kept under H₂, and micromolar amounts of sulfate, nitrate or nitrite were added. For reductant pulse experiments, small amounts of H₂-saturated KCl were added to cells incubated under N₂ with an excess of one of the above-mentioned electron acceptors. To study electron-transport driven proton translocation, the membrane potential was neutralized by addition of KSCN (100 mM). H⁺/e^- ratios of electron-transport driven proton translocation were calculated by simulation with proton.exe. This method gave lower but more realistic values than logarithmic extrapolation. We could verify the kinetic simulation parameters found with proton.exe using series of increasing additions of the reactants. Our approach allows for studying a broad variety of proton-related metabolic activities at micromolar concentrations and time scales of seconds to minutes.

Keywords: ATP synthase activity; Desulfovibrio desulfuricans; Michaelis-Menten kinetics; dissimilatory nitrate reduction to ammonia; dissimilatory sulfate reduction; proton-sulfate symport; vectorial proton translocation.

FIGURE 1

Proton-related processes in Desulfovibrio during sulfate reduction with hydrogen. (A) Electroneutral proton-sulfate symport; (B) electrogenic proton-sulfate symport; (C) release of H₂S by diffusion and partial dissociation to H⁺ + HS^–; (D) ATP synthase; (E) proton-translocating electron transport chain; and (F) periplasmic hydrogenase coupled to cytochrome c that channels electrons into the cell by vectorial electron transport. Red arrows point to electrogenic vectorial processes.

FIGURE 2

Scalar pH changes correlated with sulfate and nitrate reduction by Desulfovibrio desulfuricans CSN. A cell suspension with OD₄₃₆ = 1 was incubated at 30°C in H₂-saturated 150 mM KCl, before small pulses of 1 mM Na₂SO₄, NaNO₃, or HCl (in 150 mM N₂-flushed KCl) were added. The red dots show the simulation obtained with proton.exe (parameters see Supplementary Material, standard deviations see Supplementary Table 1). *Data from Krekeler and Cypionka, 1995. Note that a satisfying simulation was obtained only if additions of 15 nmol sulfate and 22.5 nmol H⁺ (second calibration pulse) were assumed, while the original publication says 20 nmol sulfate and 30 nmol H⁺. Even without simulation by proton exe, it is obvious that the second H⁺ pulse gave a far smaller response than the 30/40 of the first one of 40 nmol H⁺. Further sulfate and H⁺ additions in the original experiment (Krekeler and Cypionka, 1995) confirmed the scalar consumption of 1.5 H⁺ per reduced sulfate.

FIGURE 3

Sulfate reduction by Desulfovibrio desulfuricans CSN grown under sulfate limitation. Conditions as described in Figure 1, data from Cypionka (1995). Additionally to the pH curve, sulfate uptake and sulfide release were simulated by means of proton.exe (parameters see Supplementary Material, standard deviations see Supplementary Table 1). The simulation includes initial proton-sulfate symport followed by sulfate reduction and release of H₂S part of which dissociating to H⁺ + HS^–.

FIGURE 4

Oxidant pulse experiment. Proton translocation coupled to nitrate- and nitrite reduction. Nitrate-grown cells were incubated under H₂ in the presence of KSCN (100 mM). Sulfate was not metabolized under these conditions. pH curves and proton translocation were simulated by means of proton.exe (parameters see Supplementary Material, standard deviations see Supplementary Table 1). The H⁺/e^– ratio was additionally calculated by logarithmic extrapolation back to the time of electron acceptor addition (see Supplementary Figure 1).

FIGURE 5

Reductant pulse experiments. Proton translocation induced by H₂ pulses to cell suspensions incubated in N₂-saturated KCl with KSCN (100 mM) and (A) sulfate-grown cells with 10 mM Na₂SO₄ or (B) nitrate-grown cells with 10 mM NaNO₃. Three different H₂ concentrations were added for each electron acceptor. Correspondingly the simulations by proton.exe differ only by the amounts of electron donor added (parameters see Supplementary Material, standard deviations see Supplementary Table 1).