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Ion Transport and Methane Production in Methanobacterium Thermoautotrophicum FRANK D Proc. Natl. Acad. Sci. USA Vol. 91, pp. 4466-4470, May 1994 Biochemistry Ion transport and methane production in Methanobacterium thermoautotrophicum FRANK D. SAUER*t, BARBARA A. BLACKWELLO, AND JOHN K. G. KRAMER* *Centre for Food and Animal Research and *Plant Research Centre, Research Branch, Agriculture Canada, Ottawa, ON, Canada KlA 0C6 Communicated by Ralph T. Holman, January 3, 1994 (receivedfor review April 28, 1993) ABSTRACT In Methanobacterium thermoautotrophicum, contributed another 50 mV to Adi but without net HI extru- the protonmotive force for the HW-translocating ATPase con- sion. sists mainly of a transmembrane electrical gradient (A*r). These ceils do not establish a s cnt pH gradient (inside alkaline) and, in fact, if the suspending me- MATERIALS AND METHODS dium is of pH > 7.0, the pH gradient may be reversed-i.e., Measurement of Bacterial Cell Parameters. Cells of Mb. inside acid with respect to the extracellular pH. These studies thermoautotrophicum were grown and harvested as de- show by both 23Na NMR and =Na+ distribution that Na+ scribed (1, 5). Cell transfers were carried out in an anaerobic extrusion with the generation of Ad precedes methanogenesis chamber (Coy Laboratory Products, Ann Arbor, MI). Cells in Mb. thermoautotrophicum. It is calculated that the newly (-7.5 mg of protein) were incubated in 50 mM Tris/5 mM established Na+ gradients increase Ad by =50 mV (inside potassium phosphate buffer (pH 7.0) with 50 mM Tris car- negative). There is no detectable H+ extrusion during methane bonate (pH 7.0) under, H2 at 600C in a final volume of 1.5 ml synthesis; instead there is a high rate of H+ consumption for with constant shaking in 5-ml sealed rubber-stopped flasks. methane synthesis and an increase in internal pH. This was The ATP content of cells was assayed by the luciferin/ supported by 31P NMR experiments, which showed an internal luciferase assay (1, 6). pH shift from 6.8 to 7.6. With the cells maintain at an Aqd was determined by measuring the transmembrane dis- external pH of 7.2, the initial transmembrane pH gradient of tribution at equilibrium of either 86Rb+ (with 67 ,uM valino- -0.4 (inside acid) at 60VC is equivalent to Aeof +27 mV (inside positive); after 20 min of incubation, the membrane pH mycin) or tetraphenylphosphonium ion ([14C]Ph4P+), essen- gradient is +0.4 (inside alkali), which at 60°C is equivalent tially as described (7). Na+ transport was measured from the to Ad of -27 mV (inside negative). Actively respiring cells transmembrane distribution of 22Na+ as described (6). generated a protonmotive force of -198 mV. It is proposed that Cells loaded with oxidized benzyl viologen (BV2+) were energy for CO2 reduction to the level offormaldehyde (the first prepared by transferring cells (-50 mg ofprotein) into 50 mM step in methane synthesis) in Mb. thernmautotrophicum is Tris/5 mM potassium phosphate (pH 7.0) with 10 mM NaCl, derived from the Adgenerated by electrogenic Na+ extrusion. 10 mM KC1, and 5 mM BV2+ in a total volume of6.0 ml. The The protonmotive force required for ATP synthesis consists mixture was incubated in a 10-ml sealed vial for 10 mini at 600C primarily of Ad and appears to be the result of both an under H2 and chilled in ice, and the H2 replaced by Ar. These electrogenic Na+ extrusion and a pH gradient (inside alkale) cells had very active hydrogenase activity but were incapable which develops during methanogenesis. of synthesizing methane from CO2 and H2- 23Na and 31p NMR. 23Na and 31P NMR spectra were The mechanism for the generation ofprotonmotive force (Ap) recorded on a Bruker AM500 NMR spectrometer operating in methanobacteria remains controversial. There is no de- at 132.3 MHz and 202.5 MHz, respectively. 23Na NMR tectable proton translocation from inside to outside in Meth- spectra were acquired at "'600C in 10-mm NMR tubes using anobacterium thermoautotrophicum cells synthesizing meth- a 50-kHz sweep width, 900 (26-pusec) pulse, 8000 data points, ane at pH 2 7.0, but these cells are nonetheless competent to and 0.08-sec acquisition time. One thousand scans were generate ATP and establish an energy charge of 0.6 (1). collected, making the total time per spectrum 80 sec. The first Nonetheless, acid-pulse experiments indicate that ATP syn- spectrum represents an average time of 100 sec from the time thesis in Mb. thermoautotrophicum is catalyzed by a H+- the sample was inserted in the magnet. Spectra were acquired translocating ATPase (FoF1 ATPase) (2). While methanobac- in unlocked mode. Free-induction decays (FIDs) were pro- teria have been reported to translocate Na+ during metha- cessed by eliminating the first 18 points from each FID, nogenesis (3), these organisms do not appear to possess a zero-filling, and using a line broadening of4 Hz. NaCl at 100 Na+-translocating ATPase. Na+-pumping ATP synthase, mM was used as a chemical-shift standard (0 ppm). Spectra which responds specifically to Na+ but not to protons, has so were plotted in absolute intensity mode for integration. far been found only in Propionigenium modestum (4). It is The concentration of the paramagnetic-shift reagent dys- likely that methanobacteria must generate Ap required for prosium tripolyphosphate [Dy(P3010)1-] was adjusted to give ATP synthesis primarily from a transmembrane electrical a chemical-shift difference of =10 ppm between the intracel- potential (Aq*). lular and extracellular Na+ pools (8, 9). Na+ concentrations This paper confirms an earlier finding (1) that Mb. ther- were calculated by comparison ofintegrated peak intensity to moautotrophicum cells do not develop a large transmem- a resonance of 1 mM NaCl solution acquired under the same brane pH gradient (ApH) (inside alkaline) and shows that Na+ conditions. Intracellular Na+ concentrations were deter- efflux precedes the onset of methanogenesis and that this mined by correcting for average cell volume and cell con- increases Aqkby -50 mV. During methanogenesis, an internal centrations, assuming no "invisible" Na+ (10). pH shift toward alkaline was detected by 31P NMR, which Abbreviations: BV2+, oxidized benzyl viologen; TCS, 3,5,3',4'- The publication costs of this article were defrayed in part by page charge tetrachlorosalicylanilide; Ap, protonmotive force; ApH, transmem- payment. This article must therefore be hereby marked "advertisement" brane pH gradient; A*, transmembrane electrical gradient. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4466 Downloaded by guest on October 3, 2021 Biochemistry: Sauer et aL Proc. Natl. Acad. Sci. USA 91 (1994) 4467 31P NMR spectra were acquired in 10-mm tubes with 6000 There appears to be no net H+ extrusion in these cells once scans, 16,000 data points, 700 (15-,usec) pulse, 0.4-sec acqui- methane synthesis has been initiated. What happens to the sition time, and 20-kHz sweep width. Spectra were refer- internal pH is less clear. Attempts to monitor internal pH enced to 85% H3PO4 at 0 ppm. The probe was maintained at changes by radiolabeled marker distribution present techni- 40C to reduce the effects of the high concentration of para- cal difficulties, not the least of which is the maintenance of magnetic ions found in these cells. strict anaerobiosis during sampling. Changes in internal pH, however, can be measured noninvasively by 31P NMR. Fig. 2 shows the partial 31P NMR spectra of a cell suspension in RESULTS 5 mM potassium phosphate buffer before and after incubation Mb. thermoautotrophicum incubated under CO2/H2 (1:4) with Tris carbonate. The sample of Fig. 2B showed substan- showed a period of rapid H+ uptake once methane synthesis tial methane production. Before incubation, a resonance was initiated (Fig. 1). This raised the pH of the external bulk assigned to the cytoplasmic inorganic phosphate appeared at phase from 7.5 to >8.3. The pH change was equivalent to 105 2.12 ppm, corresponding to a pH of 6.8. This resonance ,umol of H+ taken up by the cells in 20 min. The addition of shifted downfield upon incubation to disappear under the TCS resulted in only a slight decrease in extracellular pH external inorganic phosphate resonance at 2.90 ppm, corre- (Fig. 1). Methane production after 30 min was 102.5 pmol. sponding to a pH of7.6. This internal pH shift of0.8 unit was These results indicate that during methanogenesis H+ flux is verified by repeating the experiment in the absence of exter- directed inward and, as was shown previously (1), there nal potassium phosphate in the buffer. Within experimental appears to be no net H+ extrusion. error, there was no shift in the external inorganic phosphate To show that the Mb. thermoautotrophicum cytoplasmic resonance. The other notable change upon incubation was membrane is impermeable to H+ and that H+ extrusion, if the appearance of a new sugar phosphate resonance at 4.0 present, would be detectable in the external bulk aqueous ppm. phase, BV2+-loaded cells were prepared. Only BV+ will In what appears to be the absence of an effective ApH cross the cytoplasmic membrane (11) as BV2+ is membrane- (inside alkaline), Mb. thermoautotrophicum depends on the impermeant. When exposed to H2, Mb. thermoautotrophi- generation of A*as the principal component of Ap. With this cum cells with 67 mM BV2+ inside rapidly reduce the in mind, the time course and magnitude of Na+ transmem- oxidized species according to reaction 1. brane gradients were measured either by cell filtration with 22Na+ or by 23Na NMR as described by Castle et al. (8, 10). H2 + 2BV2+ -- 2H+ + 2BV+ [1] 23Na NMR has been used to measure intracellular Na+ (12).
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