Institut für Chemie und Biologie des Meeres, Universität Oldenburg,D-26111 Oldenburg, Germany1
Author for correspondence: Heribert Cypionka. Tel: +49 441 798 5360. Fax: +49 441 798 3583. e-mail: Cypionka{at}icbm.de
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ABSTRACT |
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Keywords: growth yield, reversed proton gradient, sulfate transport, sodiumproton antiport, sodium ion translocation
Abbreviations: PrBCM, propylbenzylylcholine mustard.HCl; CCCP, carbonyl cyanide m-chlorophenylhydrazone; TCS, tetrachlorosalicylanilide
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INTRODUCTION |
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Alkaliphilic bacteria face the bioenergetical problem that they have to sustain an inverse transmembrane pH gradient (Krulwich, 1995 ). The intracellular pH is lower than that of their environment. In spite of this, it has been shown that some alkaliphiles can regenerate ATP coupled to proton translocation (Prowe et al., 1996
). In this case a high membrane potential must be the driving force.
In many alkaliphiles growth depends on sodium ions, which are present in high concentrations in soda lakes and most alkaline environments. Sodium ions can have several functions. They may be the coupling ions of electron transport (Tokuda & Unemoto, 1982 ). In some anaerobic bacteria sodium-ion-translocating ATPases (Heise et al., 1993
) and sodium-driven motility have been detected (Chernyak et al., 1993
). Sodium-driven ATP conservation has also been shown for another new alkaliphilic sulfate reducer, Desulfonatronum lacustre (Pikuta et al., 1998
; Pusheva et al., 1999
, 2000
). In contrast to this species, all neutrophilic sulfate reducers studied so far conserve ATP by proton translocation (Fitz & Cypionka, 1989
; Kreke & Cypionka, 1994
).
An inverse pH gradient appears less critical if energy conservation is coupled to sodium cycling. However, the cells have sodiumproton antiporters that tend to compensate for the proton versus the sodium-ion gradient (Hoffmann & Dimroth, 1991 ), and they have to regulate their intracellular pH (Krulwich et al., 1997
). Sodium ions are also symported during substrate uptake. Symport and antiport systems may function in an electroneutral or electrogenic manner, which is also of energetic relevance. In neutrophilic sulfate reducers, sodium-dependent transport of sulfate was found in marine, but usually not in freshwater, strains (Stahlmann et al., 1991
; Cypionka, 1995
). Electrogenic Na+H+ antiporters have been detected in freshwater and marine sulfate reducers (Varma et al., 1983
; Kreke & Cypionka, 1994
).
The present study was carried out in order to characterize the energy metabolism of Desulfonatronovibrio hydrogenovorans by means of physiological experiments. We used chemostats to determine growth yields and maintenance energy coefficients with sulfate and thiosulfate as electron acceptors. To study the transport of sulfate, the sodiumproton antiport mechanism and the coupling ions of chemiosmotic energy conservation, we used washed cells and chemiosmotic inhibitors.
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METHODS |
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The medium (Zhilina et al., 1997 , modified) was prepared from four stock solutions (AD) with the following final concentrations: (A) 100 mM NaCl, 2 mM NaCH3COOH, 10 mM NH4Cl, 0·5 g yeast extract l-1, 0·25 mg resazurin l-1 and 1 ml trace element solution SL9 l-1 (Tschech & Pfennig, 1984
); (B) 0·2 mM CaCl2 and 2·3 mM MgCl2; (C) 1 mM K2HPO4; and (D) 47 mM Na2CO3 and 90 mM NaHCO3. To avoid precipitations, the solutions, AD, were autoclaved separately and combined after cooling under N2. Vitamin solution (1 ml l-1) (Pfennig, 1978
) and the electron acceptor sodium sulfate (15 mmol l-1) or sodium thiosulfate (10 mmol l-1) were then added to the solution. The pH was adjusted to 9·49·6 with HCl or NaOH. The medium was reduced with small amounts of sodium dithionite until the redox indicator resazurin was decolourized.
Cells were grown in chemostats (Cypionka & Pfennig, 1986 ) with H2 as electron donor and the electron acceptor (sulfate or thiosulfate) as the limiting factor. To avoid loss of NH3 from the medium the inflowing gas was flushed through an ammonia solution (106 mM, pH 9·5).
Measurement of growth parameters.
Growth was followed by measuring the OD at 436 nm with a Shimadzu UV-1202 photometer, as well as by determination of the protein concentration according to the method of Bradford (1976) ; cell dry mass was determined according to the method of Cypionka & Pfennig (1986)
. To prove electron acceptor limitation in chemostats, ion chromatography was used to detect sulfate and thiosulfate (Fuseler et al., 1996
).
Determination of the activities of washed cells.
Cells were harvested by centrifugation of freshly grown cultures for 10 min at 15000 r.p.m. The pellet was resuspended, washed once in N2-saturated salt solution, and finally stored on ice in the same solution under H2. The composition of the salt solutions varied, as described below, depending on the type of experiment to be conducted.
Various cell activities were studied at 30 °C in a multi-electrode chamber (Cypionka, 1994 ), which allowed simultaneous measurement of O2, pH and sulfide. The reduction and disproportionation of sulfur compounds was studied with cells at a final concentration of 0·36 mg protein ml-1 in a H2-or N2-saturated salt solution with 320 mM NaCl, 80 mM KCl, 10 mM MgCl2, and 2 mM K2CO3 (pH 9·5). After preincubation for 30 min, sulfur compounds (pulses of 550 µM) were added from 10 mM stock solutions.
To analyse proton extrusion after pulses of sodium chloride, the cells were suspended in a solution of 350 mM KCl, 10 mM MgCl2 and 200 mM KSCN (pH 9·2). The permeant thiocyanate was present to destroy the membrane potential. After 30 min equilibration, sodium chloride pulses were added out of a 4 M stock solution.
Proton translocation coupled to the reduction of oxygen was measured with cells (0·4 mg protein ml-1) suspended in a H2-saturated solution of 300 mM NaCl, 20 mM KCl and 10 mM MgCl2 (pH 9·5). Small oxygen pulses were added in the form of an O2-saturated salt solution (Fitz & Cypionka, 1989 ). The chemiosmotic inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP), tetrachlorosalicylanilide (TCS), valinomycin and monensin were dissolved in methanol (1050 mM stock solutions) and applied at a concentration of 50 µM.
Estimation of intracellular pH.
The intracellular pH was studied with cells (1·1 mg protein ml-1) suspended in N2-saturated salt solution (320 mM NaCl, 80 mM KCl and 10 mM MgCl2) adjusted to different pH values by NaOH or HCl. Butanol (4%) was then added to perforate the cell membranes; pH changes were followed as described by Scholes & Mitchell (1970) .
Study of transport of various salts.
Uptake of different ions was determined by measuring the changes in light scattering in cell suspensions (Mitchell & Moyle, 1969 ; Varma et al., 1983
; Kreke & Cypionka, 1994
). Cells were suspended at OD436 1·0 (light path=1 cm) in 2 ml 175 mM KCl with 10 mM MOPS buffer (pH 7·0). After preincubation until the optical density was constant, 150 µl 5 M salt solutions (350 mM) were added and the changes in light scattering were measured with a Shimadzu UV-1202 photometer.
Measurement of the dependency of ATP production on proton or sodium ions.
Cells (0·725 mg protein ml-1) were incubated on ice in a solution of 320 mM NaCl, 80 mM KCl and 10 mM MgCl2 (pH 9·5). Twenty microlitres of 0·1 M HCl (resulting in a pH shift of 2 units), or pulses of NaCl or KCl (both 0·52 M), were then added; subsamples of 100 µl were analysed for ATP as described by Kreke & Cypionka (1994 ).
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RESULTS |
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![]() | (1) |
Ymax values for growth with sulfate and thiosulfate were 5·5 and 12·8 g cell dry mass (mol electron acceptor reduced)-1, respectively. The maintenance coefficients were 1·9 and 1·3 mmol (g dry mass)-1 h-1 for growth on sulfate and thiosulfate, respectively.
Estimation of the intracellular pH
In salt solutions with pH values between 8·0 and 9·0, cell lysis with butanol according to Scholes & Mitchell (1970) led to an acidification, whereas at pH 6·9 a slight alkalinization was observed. In control experiments without cells, butanol had no effect on the pH. These results confirmed that the cytoplasm of D. hydrogenovorans is more acidic than its environment during growth.
Reduction of different electron acceptors, and disproportionation or oxidation of sulfur compounds
Sulfate-grown washed cells reduced additions of 20 µM sulfate, sulfite and sulfur to equimolar amounts of sulfide with rates of 1060 nmol min-1 (mg protein)-1. Reduction of sulfate and sulfite was accompanied by alkalinization (disappearance of 1 H+ mol-1) and reduction of sulfur was accompanied by acidification (production of 1 H+ mol-1), as expected from the chemical reactions shown in equations (2), (3) and (5) (Table 1). Thiosulfate was reduced incompletely and only after the addition of sulfate. By contrast, thiosulfate-grown cells reduced thiosulfate without the addition of sulfate [Table 1
, equation (4)]. Sulfate reduction was completely inhibited by the uncoupler CCCP, whereas sulfite reduction was not affected.
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Reduction of sulfate and sulfite depended on the presence of sodium ions. If the cells were incubated in a salt solution without sodium ions added no formation of sulfide after pulses of sulfate or sulfite occurred. After adding NaCl (200 mM), sulfide formation started (Fig. 2). Elemental sulfur was reduced independently of sodium ions. Cells grown with thiosulfate dismutated this compound also in the absence of sodium ions. The cells were also able to oxidize sulfide with oxygen, as described in detail by Fuseler et al. (1996)
.
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Proton translocation coupled to the reduction of oxygen
A transient alkalinization of the bulk medium was observed when small oxygen pulses (5100 nmol O2, which were reduced within a few seconds) were added to cells incubated in weakly buffered H2-saturated salt solution (Fig. 5). The curves obtained were very similar to those observed during electron-transport-coupled proton translocation with neutrophilic sulfate reducers (Fitz & Cypionka, 1989
; Cypionka, 1995
, 2000
), except that an alkalinization instead of an acidification was observed. The H+:O2 ratios observed with different cell preparations were 0·21·0, independent of the pH of the assay (pH 7·59). The effect was hardly sensitive to the presence of the uncoupler CCCP (by contrast to neutrophilic sulfate reducers). Highest H+:O2 ratios (2·13·4) were obtained in the presence of valinomycin, an electrogenic K+ transporter added to dissipate the membrane potential, and monensin, an electroneutral Na+H+ antiporter, whereas either of these reagents alone had little effect. These results indicated that the pH effects might be due to electron-transport-driven sodium ion translocation coupled to Na+H+ antiport as discussed below.
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DISCUSSION |
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Sulfur metabolism
Although the spectrum of electron donors utilized by D. hydrogenovorans is very restricted (Zhilina et al., 1997 ) the bacterium reveals a versatile sulfur metabolism (Table 1
). Growth yields with thiosulfate were higher than those with sulfate. This indicates that sulfate activation requires energy, as is known from neutrophilic bacteria. Correspondingly, sulfate, but not sulfite, reduction was inhibited by uncouplers. In the absence of electron donors, disproportionation of sulfite, thiosulfate and sulfur was catalysed, but did not support growth, as found in many neutrophilic sulfate reducers (Krämer & Cypionka, 1989
). With oxygen as electron acceptor even aerobic respiration (not coupled to growth) and oxidation of sulfide were observed. The mechanism of sulfur compound disproportionation and oxidation appears to follow the same pathway in neutrophilic sulfate reducers (Fuseler et al., 1996
).
Energy coupling
It was clearly demonstrated that ATP is conserved by a proton-translocating ATPase. HCl pulses to the bulk medium resulted in ATP formation, whereas NaCl or KCl pulses showed no effect. Furthermore, ATP formation was sensitive to protonophores. The mechanism of chemiosmotic ATP synthesis in D. hydrogenovorans thus resembles that in neutrophilic sulfate reducers (Fitz & Cypionka, 1989 ), but clearly differs from that in the second species of alkaliphilic sulfate reducer Desulfonatronum lacustre, which was shown to couple ATP conservation to Na+ translocation (Pusheva et al., 2000
).
Although ATP is conserved by proton translocation, three important functions of sodium ions were found. First, sulfate is taken up by electroneutral symport with (two) sodium ions. In this regard, D. hydrogenovorans resembles marine sulfate reducers. The ability to induce an electrogenic mechanism under sulfate limitation as observed in neutrophilic sulfate reducers (Stahlmann et al., 1991 ) was not tested and can not be excluded. Second, as demonstrated in plasmolysis experiments, sodium ions were used for electrogenic antiport of protons. As in other alkaliphiles (Krulwich et al., 1997
), sodium ions are important not only for substrate uptake, but also for pH homeostasis of the cytoplasm. Furthermore, this transport system must play an essential role in the generation of the membrane potential required for H+-dependent ATP synthesis. In Desulfovibrio salexigens it was found that the electrogenic Na+H+ antiporter transports more protons than sodium ions (Kreke & Cypionka, 1994
). The same stoichiometry would help Desulfonatronovibrio hydrogenovorans to increase the membrane potential as required for proton-dependent ATP synthesis at an inverse
pH. The third and most important role of Na+ was demonstrated indirectly and can be derived from our proton translocation experiments with oxygen. Although the mechanism of energy coupling of oxygen reduction in sulfate-reducing bacteria is not fully understood (Cypionka 2000
), a transient vectorial proton extrusion coupled to ATP conservation has been shown repeatedly (Fitz & Cypionka, 1989
; Dilling & Cypionka, 1990
). However, in D. hydrogenovorans we observed a transient vectorial proton uptake instead. This certainly cannot be explained by electron-transport-driven proton uptake and an ATP synthase coupled to proton extrusion instead of uptake. First, it was shown that ATP synthesis is coupled to proton uptake. Second, electron-transport-coupled proton uptake would lower the membrane potential, which is inside-negative in all bacteria studied so far, and which must be the main driving force for ATP synthesis. Third, the transient proton uptake was not sensitive to uncouplers, although ATP synthesis was. In contrast, we observed the most pronounced effects when both valinomycin, which in the presence of K+ should destroy the membrane potential, and the Na+H+ antiporter monensin were added. Our observations are, however, in accordance with energy conservation by electron-transport-driven Na+ translocation and electrogenic Na+H+ antiport as suggested in Fig. 6
. Our data on the H+:O2 ratios are not yet robust enough to calculate stoichiometries. However, the finding that CCCP did not stimulate alkalinization of the medium is in accordance with the electrogenic antiport mechanism (more H+ than Na+ ions translocated) discussed above. Of course, the intracellular concentrations of the ions involved and the sodium- and proton-motive forces remain to be determined.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 August 2001;
revised 18 October 2001;
accepted 19 October 2001.
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