And Iron(III) in Shewanella Putrefaciens MR-1 CHARLES R
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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 MATERIALS AND METHODS substrates by bacteria is accompanied by extrusion of pro- tons to the cell exterior (39). When the substrate is in excess, Growth conditions. S. putrefaciens MR-1, originally iso- protons are translocated across the cytoplasmic membrane lated from the anaerobic sediments of Oneida Lake, New in direct proportion to the quantity of an available electron York, as a Mn(IV) reducer, has been previously described acceptor. Such proton translocation measurements (39) have (33-35). The cells were grown anaerobically under 100% N2 been used to assess bacterial respiration with a variety of at room temperature (approximately 22 to 24°C) in defined oxidants, including oxygen, nitrate, nitrite, nitrous oxide, medium (pH 7.4) (33) consisting of 9.0 mM (NH4)2SO4, 5.7 and fumarate (2, 4, 9, 14, 19, 39, 42). In addition, there is a mM K2HPO4, 3.3 mM KH2PO4, 2.0 mM NaHCO3, 1.0 mM recent report of iron respiration-driven proton translocation MgSO4, 0.49 mM CaCl2, 67.2 ,uM disodium EDTA, 56.6 ,M in several species of respiratory bacteria (40). Although H3BO3, 10.0 ,M NaCl, 5.4 ,uM FeSO4, 5.0 ,uM CoSO4, 5.0 recent studies have demonstrated that two different organ- ,uM Ni(NH4)2(SO4)2, 3.9 ,M Na2MoO4, 1.5 ,uM Na2SeO4, isms, Shewanella putrefaciens (33) (formerly Alteromonas 1.3 ,uM MnSO4, 1.0 ,uM ZnS04, 0.2 ,M CUSO4, L-arginine putrefaciens; 12, 30) and the unidentified bacterium GS-15 hydrochloride (20 ,ug mI-'), L-glutamate (20 ,g ml-'), and (26), are able to couple their anaerobic growth to the L-serine (20 jxg ml-l); the medium was supplemented with 15 reduction of manganese oxides, there are no reports demon- mM lactate as the carbon and energy source and with 2 mM strating respiratory proton translocation coupled to anaero- appropriate electron acceptor (e.g., nitrate or fumarate). bic manganese reduction. Vitamin-free Casamino Acids (0.1 g liter-1); Difco Labora- We have previously reported on the isolation and charac- tories, Detroit, Mich.) was added to stimulate the growth terization of a strain of the manganese- and iron-reducing rate. Where indicated, LB broth (pH 7.4) (31) was substi- bacterium S. putrefaciens (33-35). This strain, designated tuted for defined medium. Aerobic growth was accomplished MR-1, can couple its anaerobic growth on nonfermentable in foam-plugged 1-liter Erlenmeyer flasks, containing 500 ml carbon sources to the reduction of Mn(IV), Fe(III), and a of defined medium, on a New Brunswick Gyrotory shaker variety of other compounds (33, 35; C. R. Myers and K. H. (200 rpm) at room temperature. Nealson, in R. Frankel, ed., Iron Biominerals, in press). Cell preparations. Mid-log-phase MR-1 cells were har- These growth data suggest that MR-1 uses these compounds vested by centrifugation at 4°C and washed twice with as external electron acceptors. Ifthis is indeed the case, then anaerobic KKG buffer (100 mM potassium thiocyanate, 50 one should be able to demonstrate Mn(IV) and Fe(III) mM KCI, 1.5 mM glycylglycine, pH 7.1) (45). In preliminary respiration-dependent proton translocation in MR-1 under experiments, this concentration of potassium thiocyanate anaerobic conditions. Such data are essential to confirm that was found to be optimal for proton translocation in response these compounds are in fact used as terminal electron to 02- Washed cells were suspended in 5.0 ml of anaerobic acceptors for respiration. KKG buffer containing 2 mM lactate (as the electron donor) In this report, we present evidence for respiratory proton to a density of approximately 1 to 4 mg of total cellular translocation linked to the anaerobic reduction of Mn(IV) protein per ml. This cell density is similar to that used by and Fe(III) is S. putrefaciens MR-1. other investigators in studies of other bacteria (16, 42, 45); (A preliminary report of this work has appeared previ- for the volumes of oxidant added, variance of cell density ously [C. R. Myers and K. H. Nealson, Abstr. Annu. Meet. within this range had no effect on the observed magnitude of Am. Soc. Microbiol. 1989, 1-98, p. 233].) 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 * Corresponding author. (29, 32). Cell suspensions without added lactate usually t Present address: Department of Biology/Microbiology, South exhibited proton translocation for several hours, presumably Dakota State University, Brookings, SD 57007. through the use of endogenous electron donor (6, 40); 6232 VOL. 172, 1990 MANGANESE-IRON RESPIRATION-LINKED PROTON TRANSLOCATION 6233 nonetheless, lactate was included to provide consistent sterile fumarate-grown nitrate-grown conditions and results and to avoid potential problems that cells cells could arise by not knowing the points at which endogenous substrates transfer electrons to the respiratory chain (6, 9). P.Y' Oxidant pulse studies. The experiments were done by the KKG"buffer" oxidant pulse technique, at room temperature, as described by others (16, 40). A 5-ml sample of the washed cell suspension was placed in a reaction vessel (20-ml vial) that contained a small magnetic stir bar and was fitted with a I / 2min50 Teflon-silicone septum. A semimicro combination pH elec- fumarate V trode (Orion Ross 81-03; Orion Research Inc., Boston, Mass.) and Teflon microtubing for gas inlet and outlet were 71 50 nmol H+ inserted through the septum. With continuous stirring, the suspension was bubbled for 20 min with 02-free N2 to establish anaerobic conditions (N2 was passed through a MnO2 column of hot reduced copper filings to remove traces of oxygen). A continuous flow of 02-free N2 was blown through the headspace of the vessel for the remainder of the exper- iment. The pH of the suspension was adjusted to 7.1 with I I anaerobic HCl or KOH as necessary. 02- KKG Solutions (2.0 mM, pH 7.10) of electron acceptors in KKG buffer were prepared in gas-tight serum vials and were made anaerobic by vigorous bubbling with O2-free N2 for at least 10 min. Oxygen microelectrode measurements of solutions FIG. 1. Proton pulse traces obtained with fumarate-grown and prepared in this manner indicated that there was no detect- nitrate-grown S. putrefaciens MR-1 cells and a cell-free (sterile) able 02 present. MnO2 was prepared daily as previously control. Cells were suspended in anaerobic KKG buffer (see text) described (33), suspended to 2 mM in KKG buffer, sonicated with 2 mM lactate as the electron donor. Arrows indicate injection to reduce particle size, and set to pH 7.10 before being made of the following electron acceptors (as indicated at the left): 40 ,ul of anaerobic. Iron was prepared as ferric citrate (2 mM) in anaerobic KKG buffer, 40 p.l of a 2 mM anaerobic solution (in KKG) KKG buffer or amorphous ferric oxyhydroxide (a-FeOOH) of fumarate or MnO2, and 40 ,ul of air-saturated KKG buffer (24, 25, 34), suspended in KKG buffer; both were set to pH (02-KKG). Time and H+ calibration are as shown at the right. 7.10 before being made anaerobic. For tests with MnO2, MnO2 was never added to a cell suspension to which Fe(III) In preliminary experiments in which the permeant ion had been previously added, as the reduced iron product, triphenylmethylphosphonium bromide (40) or valinomy- Fe(II), can act as a reductant of MnO2 in a proton-liberating cin-K+ (7, 16) was used, only limited success was obtained reaction (34). For tests with 2, KKG buffer was bubbled and not with all electron acceptors; other investigators have with air to equilibrate; the oxygen content of this air- noted that certain permeant ions (e.g., valinomycin) are not saturated buffer was taken to be 0.45 ,ug-atom per ml (5, 9). suitable for demonstrating proton translocation with all Known quantities (e.g., 20 to 80 jil) of the electron electron acceptors or with all bacteria (4, 39, 42). We were acceptors were injected into the anaerobic cell suspension successful with the use of thiocyanate (SCN-) (4, 7, 14, 42) with an N2-flushed gas-tight microsyringe. Proton pulse in our experiments. In oxidant pulse studies with lactate as magnitude was shown to be directly proportional to the the electron donor and SCN- as the permeant ion, proton volume of oxidant added over this range of oxidant volumes.