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JOURNAL OF BACTERIOLOGY, JUlY 1985, p. 369-375 Vol. 163, No. 1 0021-9193/85/070369-07$02.00/0 Copyright C 1985, American Society for Microbiology Proton Translocation Coupled to Dimethyl Sulfoxide Reduction in Anaerobically Grown Escherichia coli HB101 PETER T. BILOUS AND JOEL H. WEINER* Department ofBiochemistry, University ofAlberta, Edmonton, Alberta, Canada T6G 2H7 Received 27 February 1985/Accepted 22 April 1985 Proton translocation coupled to dimethyl sulfoxide (DMSO) reduction was examined in Escherichia coli HB101 grown anaerobically on glycerol and DMSO. Rapid acidification of the medium was observed when an anaerobic suspension of cells, preincubated with glycerol, was pulsed with DMSO, methionine sulfoxide, nitrate, or trimethylamine N-oxide. The DMSO-induced acidification was sensitive to the'uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (60 ,uM) and was inhibited by the quinone analog 2-n-heptyl-4- hydroxy-quinoline-N-oxide (5.6 ,LM). Neither sodium azide nor potassium cyanide inhibited the DMSO response. An apparent -*H+/2e- ratio of 2.9 was obtained for DMSO reduction with glycerol as the reductant. Formate and H2(g), but not lactate, could serve as alternate electron donors for DMSO reduction. Cells grown anaerobically on glycerol and fumarate displayed a similar response to pulses of DMSO, methionine sulfoxide, nitrate, and trimethylamine N-oxide with either glycerol or H2(g) as the electron donor. However, fumarate pulses did not result in acidification of the suspension medium. Proton translocation coupled to DMSO reduction was also demonstrated in membrane vesicles by fluorescence quenching. The addition of DMSO to hydrogen-saturated everted membrane vesicles resulted in a carbonyl cyanide p-trifluoromethoxyphenyl- hydrazone-sensitive fluorescence quenching of quinacrine dihydrochloride. The data indicate that reduction of DMSO by E. coli is catalyzed by an anaerobic electron transport chain, resulting in the formation of a proton motive force. Escherichia coli is a facultative anaerobe capable of growth. Cells were grown anaerobically for either 36 h deriving energy for aerobic growth by oxidative phosphory- (DMSO) or 24 h (FUM) at 37°C. lation or anaerobic growth by fermentation or anaerobic Preparation of everted envelopes. Membrane vesicles were respiration. Anaerobic respiration on fumarate (FUM), ni- prepared by French pressure cell disruption as described trate, and trimethylamine N-oxide (TMAO) have been well previously (1), except that the final membrane pellet was studied (5, 8). Several organisms, including E. coli, are suspended in 1 mM HEPES (N-2-hydroxyethylpiperazine- capable of reducing dimethyl sulfoxide (DMSO) to dimethyl N'-2- ethanesulfonic acid [pH 7.5]) containing 100 mM KCI. sulfide during growth (21), and in some cases it has been Preparation of whole cells for pH measurements. Cells were shown that DMSO can serve as the terminal electron accep- grown to late-log phase (optical density at 550 nm = 1.2 + tor during anaerobic growth (11, 20). We recently reported 0.1), harvested at 3,840 x g, and washed twice with 150 mM the ability of E. coli to grow anaerobically on a minimal KCI containing 0.5 mM dithiothreitol-0.1 mM sodium medium with DMSO as the terminal electron acceptor (1). pyrophosphate (one-fifth of medium volume per wash). The Under these conditions, an inducible and membrane-bound final cell pellet was suspended in 100 mM KCI containing 25 enzyme is synthesized which catalyzes DMSO reduction. mM KSCN, 0.1 mM sodium PP1, and 50 jxg of carbonic According to the chemiosmotic hypothesis, electron flow anhydrase per ml. The cell suspension, containing 2.8 x 109 through the respiratory chain of mitochondria, chloroplasts, cells per ml, was stored on ice in 4.0-ml volumes with either or bacteria is coupled to ATP synthesis by the formation of 7 mM GLY, 7 mM sodium formate, or 2.5 mM D-(-)-lactate a proton gradient across an energy-transducing membrane as energy sources. In some experiments, hydrogen gas (12). In the present paper, we demonstrate by pH and [H2(g)] was used as an energy source. For these experi- fluorescence quenching measurements with anaerobically ments, cells were bubbled slowly with H2(g) for 0.5 h before grown E. coli that proton translocation is coupled to DMSO use. reduction. Thus, anaerobic respiration on DMSO can serve pH measurements. For measurement of extracellular pH as an energy-yielding pathway for the growth of this orga- changes, 4.0 ml of prewarmed cells (23°C) were placed in an nism. oxygen electrode vessel (model OXSDIG, Rank Bros., Bot- tisham, Cambridge, England) and equilibrated under N2(g) MATERIALS AND METHODS or H2(g) with stirring until a stable pH was attained. Air was Growth conditions. E. coli HB101 (F- hsdR hsdM pro leu excluded by a Teflon stopper which held the pH electrode gal lac thi recA rpsL) was grown anaerobically on a minimal (model GK2321C, Radiometer, Copenhagen, Denmark). medium as described previously (1), with glycerol (GLY) as Gases, N2(g) or H2(g), were introduced over the surface of the carbon and energy source. DMSO (70 mM) or sodium the cells by syringe needle ports. The pH electrode was FUM (40 mM) were used as terminal electron acceptors for connected through a pH meter (PHM84, Radiometer) to a recorder. Dissolved oxygen was monitored to ensure anaerobic * Corresponding author. conditions were achieved and maintained throughout the 369 370 BILOUS AND WEINER J. BACTERIOL. 0 A 0cn a a. 0 C2, 0 U I I I I min H+ to C a J- 0z B a -9 I-- U- I min I I min I H+ HV If FIG. 1. Proton translocation in whole cells of E. coli HB101 grown anaerobically on GLY-DMSO medium. Late-log-phase cells were harvested, washed, and suspended in 100 MnM KCI containing 25 mM KSCN, 0.1 mM sodium PP,, 50 ,ug of carbonic anhydrase per ml, and 7 mM GLY under N2(g) until a stable pH was achieved. At the indicated points, GLY (2.7 ,umol), FCCP (60 ,uM, final concentration), and 500 nmol of DMSO, methionine sulfoxide, KNO3, TMAO, or FUM were added. The vertical arrows (H+) correspond to deflections resulting from a pulse of 500 nmol of HCI. experiment. Small additions of electron donors, acceptors, quinacrine dihydrochloride were all purchased from Sigma inhibitors, and standard solutions were introduced by Chemical Co. (St. Louis, Mo.). Carbonyl cyanide p- Hamilton microsyringes (type no. 701, Hamilton Co., Reno, trifluoromethoxyphenylhydrazone (FCCP)' was obtained Nev.). The buffering capacities of the suspensions were from Aldrich Chemical Co. (Milwaukee, Wis.). determined by injecting known quantities of 50 mM HCI in 100 mM KCI. RESULTS Estimation of -*H+/2e- stoichiometries. An estimation of Proton translocation coupled to DMSO reduction. An the number of protons translocated per electron pair trans- anaerobic suspension of E. coli HB101, prepared from cells ferred from donor to acceptor (--H+/2e-) was obtaiped as grown to late log phase in GLY-DMSO medium, was follows. The point of maximum medium acidification was preincubated in the presence of excess GLY. GLY was approximated'by extrapolating the traces of the initial rate of added as the electron donor in these experiments, as endog- proton extrusion and proton reentry to the point pf inter- enous energy was insufficient to yield proton translocation section. This maximum difference in pH was quantitated by with DMSO. A final, stable pH of 6.4 to 6.6 was achieved comparison with pH changes resulting from pulses of 500 with 02(g) saturation of less than 2%. When pulsed with a nmol of HCl (10 ,ul of 50 mM HCI containing 100 mM KCI). small quantity of DMSO, rapid acidification of the extracel- The resulting value was divided by the number of moles of lular medium was recorded which slowly, but not com- electron acceptor added to the suspension to yield an'esti- pletely, dissipated (Fig. 1A). A nearly equivalent response mate for -+H+/2e-. was obtained with the DMSO analog methionine sulfoxide as Fluorescence quenching measurements. Fluorescence the electron acceptor. quenching measurements were made on hydrogen-saturated The DMSO-induced acidification was sensitive to the everted membrane vesicles under conditions described by uncoupler FCCP (60 ,uM), resulting in an immediate deple- Jones (9). Fluorescence changes of quinacrine dihydro- tion of the proton gradient'and prevention of a further chloride were measured at an excitation wavelength of 449 response to DMSO (Fig. 1A). Although 10 p,M FCCP was nm and an emission wavelength of 510 nm with a Turner sufficient to deplete the proton gradient formed, 60 ,M was spectrofluorometer (model 430, G. K. Turner Associates, required to inhibit a further response to DMSO. The above Palo Alto, Calif.). Everted membrane vesicles (2.3 mg of data indicate that the reduction of DMSO or methionine protein) were suspended in 2.5 ml of an I-2(g)-saturated sulfoxide by E. coli is coupled to an uncoupler-sensitive buffer containing 300 mM KCl, 15 mM MgCl2, and 10 mM outward translocation of protons. HEPES (pH 7.5). Quinacrine dihydrochloride (4 ,uM), sub- It was previously reported that cells grown anaerobically strates, and inhibitors were added as indicated in the figure on GLY-DMSO medium synthesized nitrate, TMAO, and legends. FUM reductases in addition to DMSO reductase activity (1). Protein determination. Protein was estimated by a sodium As shown in Fig. 1B and C, pulses of nitrate and TMAO dodecyl sulfate Lowry procedure (10) with bovine serum resulted in proton translocation. However, we have been albumin as the standard. unable to demonstrate a respQnse to FUM with these cells Chemicals and reagents. Carbonic anhydrase, 2-n-heptyl- (Fig. 1C), despite the presence of FUM reductase activity. 4-hydroxy-quinoline-N-oxide (HOQNO), HEPES, and DMSO undergoes a two-electron reduction to form VOL.

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