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JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6232-6238 Vol. 172, No. 11 0021-9193/90/116232-07$02.00/0 Copyright © 1990, American Society for Microbiology Respiration-Linked Proton Translocation Coupled to Anaerobic Reduction of Manganese(IV) and Iron(III) in Shewanella putrefaciens MR-1 CHARLES R. MYERSt* AND KENNETH H. NEALSON Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201 Received 15 February 1990/Accepted 13 August 1990 An oxidant pulse technique, with lactate as the electron donor, was used to study respiration-linked proton translocation in the manganese- and iron-reducing bacterium ShewaneUa putrefaciens MR-1. Cells grown anaerobicaUy with fumarate or nitrate as the electron acceptor translocated protons in response to manganese (IV), fumarate, or oxygen. Cells grown anaerobically with fumarate also translocated protons in response to iron(III) and thiosulfate, whereas those grown with nitrate did not. Aerobically grown cells translocated protons only in response to oxygen. Proton translocation with all electron acceptors was abolished in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (20 ,uM) and was partially to completely inhibited by the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (50 ,uM). According to the Mitchell hypothesis, the oxidation of substrates by bacteria is accompanied by extrusion of pro- tons to the cell exterior (39). When the substrate is in excess, protons are translocated across the cytoplasmic membrane in direct proportion to the quantity of an available electron acceptor. Such proton translocation measurements (39) have been used to assess bacterial respiration with a variety of oxidants, including oxygen, nitrate, nitrite, nitrous oxide, and fumarate (2, 4, 9, 14, 19, 39, 42). In addition, there is a recent report of iron respiration-driven proton translocation in several species of respiratory bacteria (40). Although recent studies have demonstrated that two different organ- isms, Shewanella putrefaciens (33) (formerly Alteromonas putrefaciens; 12, 30) and the unidentified bacterium GS-15 (26), are able to couple their anaerobic growth to the reduction of manganese oxides, there are no reports demon- strating respiratory proton translocation coupled to anaero- bic manganese reduction. We have previously reported on the isolation and charac- terization of a strain of the manganese- and iron-reducing bacterium S. putrefaciens (33-35). This strain, designated MR-1, can couple its anaerobic growth on nonfermentable carbon sources to the reduction of Mn(IV), Fe(III), and a variety of other compounds (33, 35; C. R. Myers and K. H. Nealson, in R. Frankel, ed., Iron Biominerals, in press). These growth data suggest that MR-1 uses these compounds as external electron acceptors. If this is indeed the case, then one should be able to demonstrate Mn(IV) and Fe(III) respiration-dependent proton translocation in MR-1 under anaerobic conditions. Such data are essential to confirm that these compounds are in fact used as terminal electron acceptors for respiration. In this report, we present evidence for respiratory proton translocation linked to the anaerobic reduction of Mn(IV) and Fe(III) is S. putrefaciens MR-1. (A preliminary report of this work has appeared previ- ously [C. R. Myers and K. H. Nealson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, 1-98, p. 233].) * Corresponding author. t Present address: Department of Biology/Microbiology, South Dakota State University, Brookings, SD 57007. MATERIALS AND METHODS Growth conditions. S. putrefaciens MR-1, originally iso- lated from the anaerobic sediments of Oneida Lake, New York, as a Mn(IV) reducer, has been previously described (33-35). The cells were grown anaerobically under 100% N2 at room temperature (approximately 22 to 24°C) in defined medium (pH 7.4) (33) consisting of 9.0 mM (NH4)2SO4, 5.7 mM K2HPO4, 3.3 mM KH2PO4, 2.0 mM NaHCO3, 1.0 mM MgSO4, 0.49 mM CaCl2, 67.2 ,uM disodium EDTA, 56.6 ,M H3BO3, 10.0 ,M NaCl, 5.4 ,uM FeSO4, 5.0 ,uM CoSO4, 5.0 ,uM Ni(NH4)2(SO4)2, 3.9 ,M Na2MoO4, 1.5 ,uM Na2SeO4, 1.3 ,uM MnSO4, 1.0 ,uM ZnS04, 0.2 ,M CUSO4, L-arginine hydrochloride (20 ,ug mI-'), L-glutamate (20 ,g ml-'), and L-serine (20 jxg ml-l); the medium was supplemented with 15 mM lactate as the carbon and energy source and with 2 mM appropriate electron acceptor (e.g., nitrate or fumarate). Vitamin-free Casamino Acids (0.1 g liter-1); Difco Labora- tories, Detroit, Mich.) was added to stimulate the growth rate. Where indicated, LB broth (pH 7.4) (31) was substi- tuted for defined medium. Aerobic growth was accomplished in foam-plugged 1-liter Erlenmeyer flasks, containing 500 ml of defined medium, on a New Brunswick Gyrotory shaker (200 rpm) at room temperature. Cell preparations. Mid-log-phase MR-1 cells were har- vested by centrifugation at 4°C and washed twice with anaerobic KKG buffer (100 mM potassium thiocyanate, 50 mM KCI, 1.5 mM glycylglycine, pH 7.1) (45). In preliminary experiments, this concentration of potassium thiocyanate was found to be optimal for proton translocation in response to 02- Washed cells were suspended in 5.0 ml of anaerobic KKG buffer containing 2 mM lactate (as the electron donor) to a density of approximately 1 to 4 mg of total cellular protein per ml. This cell density is similar to that used by other investigators in studies of other bacteria (16, 42, 45); for the volumes of oxidant added, variance of cell density within this range had no effect on the observed magnitude of proton translocation. Total cellular protein was determined by the method of Lowry et al. (28) on washed cell suspen- sions that had been treated at 60°C in 1 N NaOH for 15 min (29, 32). Cell suspensions without added lactate usually exhibited proton translocation for several hours, presumably through the use of endogenous electron donor (6, 40); 6232 Downloaded from https://journals.asm.org/journal/jb on 21 October 2021 by 216.165.220.226.
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Page 1: Respiration-Linked Proton Translocation Coupled to Anaerobic

JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6232-6238 Vol. 172, No. 110021-9193/90/116232-07$02.00/0Copyright © 1990, American Society for Microbiology

Respiration-Linked Proton Translocation Coupled to AnaerobicReduction of Manganese(IV) and Iron(III) in

Shewanella putrefaciens MR-1CHARLES R. MYERSt* AND KENNETH H. NEALSON

Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201

Received 15 February 1990/Accepted 13 August 1990

An oxidant pulse technique, with lactate as the electron donor, was used to study respiration-linked protontranslocation in the manganese- and iron-reducing bacterium ShewaneUa putrefaciens MR-1. Cells grownanaerobicaUy with fumarate or nitrate as the electron acceptor translocated protons in response to manganese(IV), fumarate, or oxygen. Cells grown anaerobically with fumarate also translocated protons in response toiron(III) and thiosulfate, whereas those grown with nitrate did not. Aerobically grown cells translocatedprotons only in response to oxygen. Proton translocation with all electron acceptors was abolished in thepresence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (20 ,uM) and was partially tocompletely inhibited by the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (50 ,uM).

According to the Mitchell hypothesis, the oxidation ofsubstrates by bacteria is accompanied by extrusion of pro-tons to the cell exterior (39). When the substrate is in excess,protons are translocated across the cytoplasmic membranein direct proportion to the quantity of an available electronacceptor. Such proton translocation measurements (39) havebeen used to assess bacterial respiration with a variety ofoxidants, including oxygen, nitrate, nitrite, nitrous oxide,and fumarate (2, 4, 9, 14, 19, 39, 42). In addition, there is arecent report of iron respiration-driven proton translocationin several species of respiratory bacteria (40). Althoughrecent studies have demonstrated that two different organ-isms, Shewanella putrefaciens (33) (formerly Alteromonasputrefaciens; 12, 30) and the unidentified bacterium GS-15(26), are able to couple their anaerobic growth to thereduction of manganese oxides, there are no reports demon-strating respiratory proton translocation coupled to anaero-bic manganese reduction.We have previously reported on the isolation and charac-

terization of a strain of the manganese- and iron-reducingbacterium S. putrefaciens (33-35). This strain, designatedMR-1, can couple its anaerobic growth on nonfermentablecarbon sources to the reduction of Mn(IV), Fe(III), and avariety of other compounds (33, 35; C. R. Myers and K. H.Nealson, in R. Frankel, ed., Iron Biominerals, in press).These growth data suggest that MR-1 uses these compoundsas external electron acceptors. If this is indeed the case, thenone should be able to demonstrate Mn(IV) and Fe(III)respiration-dependent proton translocation in MR-1 underanaerobic conditions. Such data are essential to confirm thatthese compounds are in fact used as terminal electronacceptors for respiration.

In this report, we present evidence for respiratory protontranslocation linked to the anaerobic reduction of Mn(IV)and Fe(III) is S. putrefaciens MR-1.(A preliminary report of this work has appeared previ-

ously [C. R. Myers and K. H. Nealson, Abstr. Annu. Meet.Am. Soc. Microbiol. 1989, 1-98, p. 233].)

* Corresponding author.t Present address: Department of Biology/Microbiology, South

Dakota State University, Brookings, SD 57007.

MATERIALS AND METHODS

Growth conditions. S. putrefaciens MR-1, originally iso-lated from the anaerobic sediments of Oneida Lake, NewYork, as a Mn(IV) reducer, has been previously described(33-35). The cells were grown anaerobically under 100% N2at room temperature (approximately 22 to 24°C) in definedmedium (pH 7.4) (33) consisting of 9.0 mM (NH4)2SO4, 5.7mM K2HPO4, 3.3 mM KH2PO4, 2.0 mM NaHCO3, 1.0 mMMgSO4, 0.49 mM CaCl2, 67.2 ,uM disodium EDTA, 56.6 ,MH3BO3, 10.0 ,M NaCl, 5.4 ,uM FeSO4, 5.0 ,uM CoSO4, 5.0,uM Ni(NH4)2(SO4)2, 3.9 ,M Na2MoO4, 1.5 ,uM Na2SeO4,1.3 ,uM MnSO4, 1.0 ,uM ZnS04, 0.2 ,M CUSO4, L-argininehydrochloride (20 ,ug mI-'), L-glutamate (20 ,g ml-'), andL-serine (20 jxg ml-l); the medium was supplemented with 15mM lactate as the carbon and energy source and with 2 mMappropriate electron acceptor (e.g., nitrate or fumarate).Vitamin-free Casamino Acids (0.1 g liter-1); Difco Labora-tories, Detroit, Mich.) was added to stimulate the growthrate. Where indicated, LB broth (pH 7.4) (31) was substi-tuted for defined medium. Aerobic growth was accomplishedin foam-plugged 1-liter Erlenmeyer flasks, containing 500 mlof defined medium, on a New Brunswick Gyrotory shaker(200 rpm) at room temperature.

Cell preparations. Mid-log-phase MR-1 cells were har-vested by centrifugation at 4°C and washed twice withanaerobic KKG buffer (100 mM potassium thiocyanate, 50mM KCI, 1.5 mM glycylglycine, pH 7.1) (45). In preliminaryexperiments, this concentration of potassium thiocyanatewas found to be optimal for proton translocation in responseto 02- Washed cells were suspended in 5.0 ml of anaerobicKKG buffer containing 2 mM lactate (as the electron donor)to a density of approximately 1 to 4 mg of total cellularprotein per ml. This cell density is similar to that used byother investigators in studies of other bacteria (16, 42, 45);for the volumes of oxidant added, variance of cell densitywithin this range had no effect on the observed magnitude ofproton translocation. Total cellular protein was determinedby the method of Lowry et al. (28) on washed cell suspen-sions that had been treated at 60°C in 1 N NaOH for 15 min(29, 32). Cell suspensions without added lactate usuallyexhibited proton translocation for several hours, presumablythrough the use of endogenous electron donor (6, 40);

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MANGANESE-IRON RESPIRATION-LINKED PROTON TRANSLOCATION 6233

nonetheless, lactate was included to provide consistentconditions and results and to avoid potential problems thatcould arise by not knowing the points at which endogenoussubstrates transfer electrons to the respiratory chain (6, 9).Oxidant pulse studies. The experiments were done by the

oxidant pulse technique, at room temperature, as describedby others (16, 40). A 5-ml sample of the washed cellsuspension was placed in a reaction vessel (20-ml vial) thatcontained a small magnetic stir bar and was fitted with aTeflon-silicone septum. A semimicro combination pH elec-trode (Orion Ross 81-03; Orion Research Inc., Boston,Mass.) and Teflon microtubing for gas inlet and outlet wereinserted through the septum. With continuous stirring, thesuspension was bubbled for 20 min with 02-free N2 toestablish anaerobic conditions (N2 was passed through acolumn of hot reduced copper filings to remove traces ofoxygen). A continuous flow of 02-free N2 was blown throughthe headspace of the vessel for the remainder of the exper-iment. The pH of the suspension was adjusted to 7.1 withanaerobic HCl or KOH as necessary.

Solutions (2.0 mM, pH 7.10) of electron acceptors in KKGbuffer were prepared in gas-tight serum vials and were madeanaerobic by vigorous bubbling with O2-free N2 for at least10 min. Oxygen microelectrode measurements of solutionsprepared in this manner indicated that there was no detect-able 02 present. MnO2 was prepared daily as previouslydescribed (33), suspended to 2 mM in KKG buffer, sonicatedto reduce particle size, and set to pH 7.10 before being madeanaerobic. Iron was prepared as ferric citrate (2 mM) inKKG buffer or amorphous ferric oxyhydroxide (a-FeOOH)(24, 25, 34), suspended in KKG buffer; both were set to pH7.10 before being made anaerobic. For tests with MnO2,MnO2 was never added to a cell suspension to which Fe(III)had been previously added, as the reduced iron product,Fe(II), can act as a reductant of MnO2 in a proton-liberatingreaction (34). For tests with 2, KKG buffer was bubbledwith air to equilibrate; the oxygen content of this air-saturated buffer was taken to be 0.45 ,ug-atom per ml (5, 9).Known quantities (e.g., 20 to 80 jil) of the electron

acceptors were injected into the anaerobic cell suspensionwith an N2-flushed gas-tight microsyringe. Proton pulsemagnitude was shown to be directly proportional to thevolume of oxidant added over this range of oxidant volumes.Proton pulses were recorded by using an Orion SA720 pHmeter coupled to a Hewlett-Packard 3390A integrator set to2 mV full scale. Pulse amplitudes were quantified by thegraphic methods of Scholes and Mitchell (39). On the plot ofpH versus time, the decay phase of the proton pulse wasextrapolated back to the time at which half of the maximalpulse had been achieved; the extrapolated value at this timeis considered to be an estimate of the true value, whereas therecorded maximum is considered to be an underestimate dueto the simultaneous leakage of translocated protons backinto the cytoplasm (39). Calibration was made by injectingknown quantities of anaerobic 5 mM HCl-150 mM KCl(prepared from a titrated HCl standard).

RESULTS

Anaerobic growth of S. putrefaciens MR-1 with Mn(IV) orFe(III) has been previously reported (27, 33; Myers andNealson, in press). However, such studies do not provideunequivocal evidence for energy generation from Mn and Fereduction. Studies were therefore conducted to examinerespiratory proton translocation linked to these metal oxidesin this bacterium.

sterile fumarate-grown nitrate-growncells cells

KKG "buffer" Y'P.

Ifumarate V

02- KKG

/ 2min50

71 50 nmol H+

I I

FIG. 1. Proton pulse traces obtained with fumarate-grown andnitrate-grown S. putrefaciens MR-1 cells and a cell-free (sterile)control. Cells were suspended in anaerobic KKG buffer (see text)with 2 mM lactate as the electron donor. Arrows indicate injectionof the following electron acceptors (as indicated at the left): 40 ,ul ofanaerobic KKG buffer, 40 p.l of a 2 mM anaerobic solution (in KKG)of fumarate or MnO2, and 40 ,ul of air-saturated KKG buffer(02-KKG). Time and H+ calibration are as shown at the right.

In preliminary experiments in which the permeant iontriphenylmethylphosphonium bromide (40) or valinomy-cin-K+ (7, 16) was used, only limited success was obtainedand not with all electron acceptors; other investigators havenoted that certain permeant ions (e.g., valinomycin) are notsuitable for demonstrating proton translocation with allelectron acceptors or with all bacteria (4, 39, 42). We weresuccessful with the use of thiocyanate (SCN-) (4, 7, 14, 42)in our experiments. In oxidant pulse studies with lactate asthe electron donor and SCN- as the permeant ion, protontranslocation in fumarate-grown cells was noted in responseto fumarate, MnO2, and 02 (Fig. 1). Cells grown with nitrateas the electron acceptor were likewise able to translocateprotons in response to fumarate, MnO2, and 02 (Fig. 1). Incontrol experiments, no acidification pulses were noted uponinjection of these electron acceptors into sterile KKG, norwere pulses noted upon injection of anaerobic KKG into cellsuspensions (Fig. 1).For fumarate-grown cells, we also demonstrated proton

translocation in response to nitrate and to Fe(III) citrate, asoluble form of Fe(III) (Fig. 2). As a control for the Fe(III)citrate, no acidification pulses were noted upon injection ofpotassium citrate (data not shown). No proton pulses werenoted in response to another form of Fe(III), amorphousFeOOH. However, this insoluble form of Fe(III) exists aslarge particulate clumps; in previous experiments, it wasnoted that the rate at which MR-1 reduces FeOOH ismarkedly less than the rate for more soluble forms of Fe(III)(34); Lovley and Phillips (24, 25) have also demonstrateddifferent rates of reduction for different forms of Fe(III). Itseems likely, therefore, that the lack of proton translocationin response to FeOOH results from a reduction that is tooslow to elicit a measurable proton pulse. Sonication wasineffective in reducing the particle size of these FeOOH

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6234 MYERS AND NEALSON

KKG "buffer" >

fumarate

Fe(IIl) citrate I

nitrate I

O2 KKG

oH2min

50nml H+

I/

FIG. 2. Proton pulse traces obtained with fumarate-grown S.putrefaciens MR-1 cells in the presence and absence of the proto-nophore CCCP (20 ,tM). Cells were suspended in anaerobic KKGbuffer with 2 mM lactate as the electron donor. Arrows indicateinjection of the following electron acceptors (as indicated at the left):40 ,ul of anaerobic KKG buffer, 40 RI of a 2 mM anaerobic solution(in KKG) of fumarate or Fe(III) citrate or nitrate, and 40 p.l ofair-saturated KKG buffer (02-KKG). No acidification was notedupon injection of these electron acceptors into cell-free KKG buffer(data not shown). The proton pulse in response to MnO2 was alsoabolished in the presence of 20 puM CCCP (not shown). Time and H+calibration are as shown at the right.

preparations. Particle size effects of MnO2 on proton trans-location were also noted: when unsonicated or poorly soni-cated MnO2 preparations (with visibly clumped MnO2) wereused, no proton translocation was observed (data notshown). Proton pulses were observed only in response toadequately sonicated suspensions in which the MnO2 sus-

pension was very fine (i.e., there were no visible clumps).Sonicating the particles to reduce their size increases theirsurface/volume ratio, thus increasing the amount of surface-exposed MnO2 available for immediate reduction (see dis-cussion below).Proton translocation by MR-1 was abolished by addition

of the protonophore carbonyl cyanide m-chlorophenylhydra-zone (CCCP; 20 p,M), regardless of the electron acceptor(Fig. 2). This result is expected if the proton pulses seenreally were the result of the establishment of a protongradient across the membrane. In the presence of CCCP,rapid alkalinization occurred (Fig. 2), indicating that theexternal pH of 7.1 was acidic with respect to the cell interior.When the culture was allowed to equilibrate in the presenceof CCCP, an internal cell pH of approximately 8 wassuggested. Other investigators have noted comparable rapidalkalinization phases in the presence of CCCP (16). Whereasthe uncoupling agent dinitrophenol did not affect protontranslocation at a concentration of 100 p,M, 600 p,M dinitro-phenol did cause both a marked decrease in the magnitude ofthe proton pulses and a faster decay of the pulses.Because of the remarkable respiratory versatility of MR-1

(33, 35), it was possible to examine the proton translocationversatility of this bacterium when it was grown under avariety of conditions. These results, showing the numbers ofprotons translocated per two electrons transferred (-*H+/2e-), are summarized in Table 1. As described above inmore detail, cells grown anaerobically with fumarate as theelectron acceptor translocated protons in response to avariety of electron acceptors. Nitrate-grown cells exhibitedsimilar properties except that they did not exhibit a responseto Fe(III) or thiosulfate. Whereas a response to Fe(III) wasshown for nitrate-grown cells in a preliminary experiment,no such response was noted in three independent repeatexperiments (Table 1). In a preliminary report (Myers andNealson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989), wealso noted that nitrate-grown cells appeared less proficientthan fumarate-grown cells at translocating protons in re-sponse to MnO2. With the additional data now in hand,however, we see that this is not the case; although the mean-*H+/2e- ratio in response to MnO2 was slightly smaller fornitrate-grown cells than for fumarate-grown cells (Table 1),there was no significant statistical difference between thesevalues when the data were analyzed by the two-sample t test(8) at the 0.05 significance level.

Cells grown anaerobically in LB medium, a complexmedium in which the electron acceptor(s) is not known,exhibited proton pulses only in response to °2 and fumarate(Table 1). Aerobically grown cells translocated protons onlyin response to 02 (Table 1), suggesting that the anaerobicelectron transport systems are not synthesized under aerobicconditions.The demonstration of proton translocation in response to

Mn and Fe suggests that the reduction of these metals byMR-1 is likely to be linked to a conventional membrane-bound respiratory electron transport chain. Studies weretherefore done to examine the effects of certain electrontransport inhibitors. 2-Heptyl-4-hydroxyquinoline N-oxide

TABLE 1. Effects of cell growth conditions on proton translocation by MR-1

Cell Growth Conditions -*H+/2e- ratio (mean ± SD) in response toa:

Medium Electron Atmosphere 02 Fumarate MnO2 Fe(Ill) Nitrate Thiosulfateacceptor Ams

Defined Fumarate 100% N2 2.20 + 0.75 (11) 0.47 ± 0.19 (6) 0.14 ± 0.04 (5) 0.57 ± 0.12 (6) 0.16 + 0.01 (5) 0.15 ± 0.08 (4)Defined Nitrate 100%0 N2 2.36 ± 0.85 (3) 0.44 ± 0.04 (2) 0.10 ± 0.04 (3) -(3) NT (3)LB ? 100% N2 2.75 (1) 0.42 (1) -(2) NT -(2) (2)Defined Oxygen Air 2.79 + 0.56 (8) - (4) -(4) - (4) - (4) NT

a Numbers in parentheses represent the number of independent trials. Lactate (2 mM) was included in all experiments as the electron donor. -, No acidificationdetected; NT, not tested.

fumarate-grown cellsno CCCP +20 mM CCCP

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fumarate-grown cellsno HQNO +50 mM HQNO

°2- KKG

fumarate

2 min

MnO2 50 nmol H+

nitrate

molybdate

FIG. 3. Proton pulse traces obtained with fumarate-grown S.

putrefaciens MR-1 cells in the presence and absence of HQNO (50,M). Cells were suspended in anaerobic KKG buffer with 2 mMlactate as the electron donor. Arrows indicate injection of thefollowing electron acceptors (as indicated at the left): 40 ,u ofair-saturated KKG buffer (02-KKG), 40 ,u of a 2 mM anaerobicsolution (in KKG) of fumarate, MnO2, nitrate, and molybdate. Noacidification was noted upon injection of these electron acceptorsinto cell-free KKG buffer (data not shown). No acidification wasnoted upon injection of 40 pJ of anaerobic KKG buffer into cellsuspensions or cell-free KKG buffer (data not shown). Time and H+calibration are as shown at the right.

(HQNO; 50 ,uM) completely inhibited proton translocationin response to anaerobic electron acceptors, including fuma-rate, Mn(IV), and nitrate (Fig. 3). For cells grown anaerobi-cally with fumarate as the electron acceptor, HQNO causedonly a partial (ca. 30 to 35%) inhibition of proton transloca-tion in response to 02 (Fig. 3). Curiously, for cells grownanaerobically in LB medium, HQNO completely abolished02-linked proton translocation. Proton translocation was notobserved in response to molybdate (Fig. 3) or sulfate (datanot shown); this finding is not surprising, since MR-1 isunable to use these compounds as electron acceptors forgrowth (33, 35). The following observations were madeduring studies with other electron transport inhibitors: (i) ina single experiment, antimycin A (50 ,uM) completely inhib-ited proton pulses in response to all anaerobic electronacceptors but did not inhibit proton pumping in response to02, (ii) rotenone (40 ,uM) did not inhibit proton translocationin response to 02 or any of the anaerobic electron acceptors,and (iii) cyanide (200 ,uM) did not inhibit proton transloca-tion by aerobically or anaerobically grown cells. Consistentwith these observations, we previously reported that HQNOand antimycin A act as effective inhibitors of Mn reductionby MR-1, whereas cyanide is noninhibitory (33).While one might argue the possibility that trace levels of

contaminating 02 in the MnO2 and Fe(III) preparationscould be causing the proton pulses seen in fumarate-growncells, there are strong arguments against this view. First, all

solutions of electron acceptors were prepared in an identicalmanner by vigorous gassing with O2-free N2, yet protontranslocation was consistently seen in response to MnO2 andFe(III) but was never observed in response to sulfate andmolybdate, electron acceptors which cannot be used forgrowth by MR-1. Proton translocation was also never ob-served during numerous injections of anaerobic KKG buffer(Fig. 1). Trace amounts of 02 contamination would not haveconsistently occurred in MnO2 and Fe(III) solutions andnever in these other negative control solutions. Second, thesame N2 gas used to deoxygenate the electron acceptors wascontinuously blown through the headspace of the experi-mental vessel throughout the course of the experiments; thiscontinual flushing should have eliminated any potential 02leakage through the septum during injection; any suchproblems would have nonetheless occurred randomly andnot been confined to MnO2 and Fe(III) solutions only. Third,the results for cells grown anaerobically in LB medium oraerobically in defined medium argue against 02 contamina-tion of the MnO2 and Fe(III) preparations. These cells wereequally, or even more, responsive to 02 relative to fumarate-grown cells (Table 1), yet they did not exhibit protontranslocation in response to MnO2 or Fe(III). If 02 was infact present in the Mn(IV) and Fe(III) suspensions, then Mn-and Fe-linked proton translocation would have been seenwith these LB-grown or aerobically grown cells. Fourth, thedata presented on the effects of HQNO (Fig. 3) also argueagainst 02 contamination of the Mn(IV) and Fe(III). WhileHQNO had only a minimal effect on proton translocation inresponse to 02, it completely abolished proton pulses inresponse to anaerobic electron acceptors. This would not bethe case if these anaerobic acceptors were indeed 02 con-taminated.

DISCUSSION

MR-1 cells grown anaerobically with fumarate as theelectron acceptor translocated protons in response to 02,nitrate, MnO2, Fe(III), fumarate, and thiosulfate; MR-1 isable to couple its growth to the reduction of all of theseelectron acceptors (33, 35). In contrast, MR-1 did nottranslocate protons in response to sulfate or molybdate,electron acceptors which it cannot use for growth (33, 35).Cells grown under other conditions exhibited different re-sponse patterns to the various electron acceptors. For ex-ample, aerobically grown cells translocated protons only inresponse to 02, suggesting that 02 inhibits expression of theanaerobic respiratory components; these results are inagreement with earlier findings on S. putrefaciens whichdemonstrated that atmospheric levels Of 02 inhibit the re-duction of these anaerobic electron acceptors (33, 35). Inother bacteria, such as Escherichia coli (23) and Micrococ-cus denitrificans (20), 02 also inhibits expression of theanaerobic respiratory systems.

Nitrate-grown MR-1 cells were unable to translocate pro-tons in response to Fe(III) or thiosulfate, suggesting thatnitrate inhibits the expression or function of the compo-nent(s) necessary for the reduction of Fe(III) and thiosulfate.An alternative explanation is that nitrite, the product ofnitrate reduction, could be the inhibitory compound.Obuekwe et al. (37) demonstrated that nitrate inhibited thenet rate of Fe(III) reduction in a bacterium isolated fromcrude oil (now known to be a strain of S. putrefaciens), butsuggested that the inhibition was due to the chemical oxida-tion of Fe(II) by nitrite and not to an actual inhibition of Fereduction by nitrate. Our ability to demonstrate differential

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expression of oxidant-specific respiratory systems underdifferent growth conditions (Table 1) suggests that theseprocesses are mediated by separate and distinct reductasecomponents.An attempt was made to examine proton translocation in

MR-1 cells grown anaerobically with MnO2 as the electronacceptor. However, because MnO2 is a solid, the cellscannot be washed free of this electron acceptor, a necessarystep in preparation for the proton translocation experiments.To rid the cells of MnO2, a strong reductant (e.g., ascorbateor hydroxylamine) had to be added to the cells to reduce andsolubilize the Mn before washing; these reductants, how-ever, damaged the cells sufficiently that proton translocationwas not observed with any of the electron acceptors.With specific regard to Fe(III) reduction, Arnold et al. (1)

suggested that Pseudomonas sp. strain 200 (now known tobe a strain of S. putrefaciens) contained both a constitutive(expressed under high 02) and a low-02-inducible Fe(III)reductase. The rates of Fe(III) reduction for low-02-growncells of this strain were severalfold higher than the rates inhigh-02-grown cells. Furthermore, respiratory inhibitorswere much less effective at inhibiting Fe(III) reduction in thelow-02-grown cells than in the high-02-grown cells (1). Onthe basis of these results, electron transport to the constitu-tive Fe(III) reductase was proposed to involve quinones andcytochromes and to result in respiratory proton transloca-tion. On the other hand, the low-02-induced form of Fereductase was proposed to be coupled to an abbreviatedelectron transport chain and not to involve proton translo-cation (1). Our results (Table 1) are in conflict with these;i.e., we observed respiratory-linked proton translocation inresponse to Fe(III) in anaerobically grown cells but not inaerobically grown cells. However, both the bacterial strainand the growth conditions differed from those used byArnold et al. (1), which may have contributed to the differ-ence in results.The effects of various inhibitors on the Mn- and Fe-linked

proton translocation properties of MR-1 strengthen the im-plication that these cell-mediated reductions are linked toconventional membrane-bound respiratory processes. Twoagents which increase the permeability of membranes toprotons, CCCP and dinitrophenol, inhibited or abolished theacid pulses observed with all electron acceptors; that theacidification phases were sensitive to these agents indicatesthat they were in fact due to the establishment of a protongradient across the membrane. The inhibitory effects ofHQNO and antimycin A suggest the involvement of b- andc-type cytochromes (7, 41) in the electron transport chains.In previous studies of S. putrefaciens by others, HQNOinhibited both nitrate and Fe(III) reduction (37), and re-duced-minus-oxidized cytochrome difference spectra wereconsistent with the presence of b- and c-type cytochromes(1, 36).Both HQNO and antimycin A inhibited proton transloca-

tion in response to anaerobic electron acceptors but exhib-ited little or no effect on 02-linked proton translocation; thisfinding indicates a marked difference between the aerobicand anaerobic electron transport chains. That rotenone didnot affect proton translocation suggests the lack of partici-pation of NADH dehydrogenase in this process (41); thisresult is not surprising, since lactate was the electron donorfor both cell growth and the proton translocation experi-ments. In fact, in experiments in which cell suspensions(lactate and fumarate grown) were starved of endogenouselectron donor(s) in the presence of excess electron acceptor(e.g., fumarate) and then pulsed with potential electron

donors, only lactate was used as an electron donor forproton translocation; other potential electron donors (27, 35;Myers and Nealson, in press) tested (including pyruvate,formate, serine, and H2) did not elicit proton pulses.

Values for the -*H+/2e- quotients for various oxidantshave been reported for several other bacteria. For oxygen,with endogenous substrates as the electron donor, the valuesapproximate 4 to 8 mol of H+ translocated per mol of 0consumed and are somewhat dependent on the specificrespiratory chain composition of a given bacterium (14).Differences are also noted between independent investiga-tors for a given bacterium, with --H+/O values for Paracoc-cus denitrificans (with endogenous electron donors) of 4(21), 6 (2), and 8 (17) being reported; some of these differ-ences are no doubt the result of various procedural differ-ences (e.g., different permeant ions). While the MR-1 --H+/2e- values for oxygen of 2.2 to 2.8 (Table 1) seem low incomparison, they are similar to the value of 1.9 reported forE. coli with lactate as the electron donor (9).

Reports of --H+/2e- ratios with nitrate as the oxidantinclude values of 4.3 for P. denitrificans (17), 3.7 for Alcali-genes sp. (4), and 1.9 for E. coli (9). The last value providesa more useful comparison, since lactate was used as theelectron donor in those experiments. The value we report forMR-1 with nitrate of 0.16 is very low. This is not necessarilysurprising, however, given our use of SCN- as the permeantion. SCN- is inhibitory to nitrate reductases of other bacte-ria (4, 22, 38, 44); that SCN- can act as a metal-chelatingagent for various metals, including molybdenum, probablyexplains its inhibitory effects on nitrate reductase (38). Weused SCN- in our experiments, however, because it was thepermeant ion that gave us the best results with both Mn(IV)and Fe(III), the two oxidants in which we were mostinterested. SCN-, when compared with valinomycin-K+ ortriphenylmethylphosphonium bromide, however, has beenreported to provide the best ->H+/2e- values for oxygen inother bacteria (2, 4, 39).Our MR-1 -*H+/2e- values for fumarate of 0.47 ± 0.19 are

lower than those of 1.1 to 1.3 reported in Vibrio succino-genes (19) and 1.85 in E. coli (15). However, other reportsfor fumarate in E. coli cite suboptimal values (3, 10); suchlow values are considered to be underestimates becausefumarate transport is relatively slow and rate limiting (10). Iffumarate transport is likewise rate limiting in MR-1, ourvalues for fumarate may be underestimates of the truestoichiometry. Since membrane-linked fumarate reductionhas been found only in bacteria with either menaquinone ordesmethylmenaquinone (18, 19), the anaerobic reduction offumarate by MR-1 implies the participation of menaquinonein the anaerobic electron transport chain. Itoh et al. (13)have reported the presence of methylmenaquinone-7 in thetype strain of this bacterium, S. putrefaciens ATCC 8071(IAM 12079).

It is difficult at this point to assess how close the MR-1-+H+/2e- values for MnO2 approximate the true stoichiom-etry. It must be remembered that MnO2 is a solid substrate;therefore, it is likely that its reduction in these experimentsis rate limiting and that reduction rates will obey complexkinetics of surface chemical reactions (43). Not only will thechemistry and three-dimensional structure of the exposedsurface groups differ from those of the solid interior (43), butthe surface-exposed Mn(IV) will be what is accessible forimmediate reduction. Reduction of Mn(IV) groups in theinner portion of the particles will occur only after the outerlayers are reduced and solubilized. Only that Mn(IV) re-duced within the first few seconds will contribute to the

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MANGANESE-IRON RESPIRATION-LINKED PROTON TRANSLOCATION 6237

proton pulse, and it seems very unlikely that these relativelylarge MnO2 particles (>99% are retained by 0.22-,um-pore-size filters) are completely reduced within the first fewseconds. Unfortunately, there is no technology available toaccurately measure the amount of Mn reduced coincidentwith the proton pulse. We must therefore calculate our-)H+/2e- values for MnO2 on the basis of electron equiva-lents added. If we assume the likely possibility that not allMnO2 is immediately reduced, then the -*H+/2e- valuesreported in Table 1 are certainly underestimates of the truestoichiometry.

It is also important to keep in mind that the reduction ofMnO2 is a proton-consuming reaction: MnO2 + 2e- + 4H+-* Mn2+ + 2H20. Since it is very likely that the large size ofthe MnO2 particles requires that they be reduced extracel-lularly, this proton consumption would certainly reduce thesize of the observed proton translocation pulse. Once again,however, since we cannot measure Mn reduction coincidentwith proton translocation, we cannot correct for the numberof protons that would be consumed during Mn reduction.Surface adsorption of protons to unoccupied surface sites ofunreduced MnO2 particles (43) would also reduce the size ofthe proton pulses. Without knowing the biochemical natureof the Mn reductase component(s) or having an in vitro assayfor Mn reductase, it is also difficult to assess the potentialinhibitory effects of SCN- on the kinetics of this process. Itis also possible that certain, as yet uncharacterized cellularinteractions result in the inaccessibility of some of thetranslocated protons to the bulk phase which is in equilib-rium with the measuring pH electrode (11); such interactionscould also contribute to the observed -*H+/2e- values forMnO2 and the other electron acceptors. Given all of thisinformation, it seems almost certain that the values in Table1 for MnO2 are underestimates of the true stoichiometry.

It is likewise difficult to fully interpret the Fe(III) protontranslocation data for MR-1. Although we used a solubleform of Fe(III), we do not know the details of the reductaseitself and therefore cannot assess the possibilities of irontransport being rate limiting or SCN- being inhibitory. Weare not aware of previous reports of thiosulfate-linked pro-ton translocation, and comparison with previous reports istherefore not possible.

Regardless of the stoichiometries, however, MR-1 isclearly able to translocate protons in response to MnO2 andFe(III) and is therefore presumably capable of generating aproton motive force via the dissimilatory reduction of Mn orFe oxides. In conjunction with the growth data reportedelsewhere (33; Myers and Nealson, in press), these protontranslocation data provide unequivocal evidence for energygeneration linked to Mn(IV) and Fe(III) respiration. Thismakes MR-1 the only organism to date for which both energygeneration and growth have been linked to Mn and Fereduction. In anaerobic environments containing Mn or Feoxides (e.g., sediments and anaerobic soils), an organismsuch as MR-1 which can gain energy from the use of Mn(IV)or Fe(III) as an electron acceptor will have a distinctadvantage over those organisms for which Mn and Fereduction are non-energy-yielding processes. Studies con-cerning the biochemistry of Mn and Fe reduction by MR-1are under way. These studies should eventually lead to abetter understanding of this novel form of microbial metab-olism.

ACKNOWLEDGMENTSThis work was supported by National Science Foundation grant

NAGW-1047 and National Aeronautics and Space Administration

grant 8609778 to K. H. Nealson. C. R. Myers was supported in partby Public Health Service toxicology training grant ES07043 fromthe National Institute of Environmental Health Science.We thank B. Wimpee for the drawings.

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13. Itoh, T., H. Funabashi, Y. Katayama-Fujimura, S. Iwasaki, andH. Kuraishi. 1985. Structure of methylmenaquinone-7 isolatedfrom Alteromonas putrefaciens IAM 12079. Biochim. Biophys.Acta 840:51-55.

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