Archives of

Hicrobiology

Arch Microbiol (1989) 152: 237- 243

9 Springer-Verlag 1989

Characterization of sulfate transport in Desulfovibrio desulfuricans Heribert Cypionka Fakultfit fiir Biologie, Universitgt Konstanz, D-7750 Konstanz, Federal Republic of Germany

Abstract. Uptake of 3SS-labelled sulfate was studied with a new isolate of Desulfovibrio desulfuricans, strain CSN. Micromolar additions of sulfate ( 1 - 1 0 gM or nmol/mg protein) to cell suspensions incubated in 150 mM KC1 at -I~ were almost completely taken up and accumulated about 5,000-fold. Accumulation was not influenced by incubation in NaC1 instead of KC1, by acidic pH (5.5) or by incubation under air for 10 min. In alkaline milieu (pH 8.5), after prolonged contact with air (2 h), or after growth with excess sulfate or thiosulfate as electron acceptor, the amount taken up was diminished approximately by half. Pasteurization inhibited sulfate uptake completely. With increasing concentrations of added sulfate (0.1 to 2.5 mM) the intracellular concentration increased only slowly up to 25 mM, and the accumulation factor decreased down to 8. Sulfate transport was reversible. Accumulated sulfate was rapidly lost from the cells after addition of excess non-labelled sulfate or after addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ATPase inhibitor dicyclohexylcarbodiimide (DCCD) specifically inhibited sulfate reduction but had no immediate influence on sulfate accumulation. Addition of the phosphate analogue arsenate (5 mM) was without effect. These results were not in favour of an ATP-dependent transport system. The K +-H +-antiporter nigericin (in 150 m M KC1) and the Na +-H +-antiporter monensin (in 150 m M NaC1) caused partial inhibition of sulfate accumulation, whereas the K+-transporter valinomycin (in 150 mM KC1) and the N a + - H + exchange inhibitor amiloride (2 mM) were without effect. The permeant thiocyanate anion (150 mM) inhibited sulfate uptake by 60% at pH 7, and completely at pH 8.5. Although the effects of the different ionophores on the chemiosmotic gradients have not been studied so far, the results indicated that probably both, ApH and A ku drive sulfate accumulation and that sulfate is taken up electrogenically in symport with more than 2 protons. The structural sulfate analogues tungstate and molybdate (0.1 mM, each) did not affect sulfate accumulation, although molybdate inhibited sulfate reduction. Chromate completely blocked both of these activities. Sulfite and selenite caused little or no decrease of sulfate accumulation, whereas with thiosulfate and selenate significant inhibition was observed.

Sulfate-reducing bacteria conserve energy from the reduction of sulfate to H2S. This process has only a low free energy change, and sulfate must be activated by means of ATP prior to reduction. Due to the ATP requirement for its activation, sulfate must be taken up through the cytoplasmic membrane. In some bacteria like Salmonella typhimurium or Escherichia coli, that use sulfate only for assimilation of cellular sulfur compounds, sulfate transport is driven by hydrolysis of ATP (Furlong 1987). For dissimilatory sulfatereducing bacteria such an additional ATP requirement would possibly make net energy conservation impossible. In a recent study (Cypionka 1987), it was reported that in Desulfovibrio desulfuricans sulfate is taken up in symport with protons. During uptake of sulfate in a non-buffered medium, the disappearance of two protons was observed. This might be interpreted as a hint on electroneutral symport of two protons with the double-charged sulfate anion. Electroneutral sulfate transport (uptake as H2SO4) would be advantageous for the cells, as the gaseous end-product of sulfate reduction, HzS, would remove the protons taken up together with sulfate by simple diffusion out of the cell. Thus, electroneutral sulfate transport would not consume net energy during growth. However, the cells are able to accumulate micromolar sulfate additions more than 5,000fold. For this accumulation, a reversible electroneutral proton-anion symport mechanism would require a ApH of about 2 (i. e. Log[accumulation] = 2 ApH, inside alkaline), if sulfate would not be withdrawn from equilibrium by further reactions. The experiments described so far (Cypionka 1987) mainly included observations on proton movements and sulfide production under various conditions. In the present study, radiolabelled sulfate was used to characterize sulfate transport. Sulfate accumulation was found to be reversible and probably dependent on both, ApH and A~. It is proposed that sulfate uptake at low sulfate concentrations is electrogenic, consuming more than 2 protons per sulfate and net energy during growth.

Key words. Desulfovibrio desulfuricans - Dissimilatory sulfate reduction - Sulfate transport -- Electrogenic proton-anion symport - Sulfate analogues

As the susceptibility of Desulfovibrio desulfuricans strain Essex to various ionohores was found to be low (Cypionka 1987), a newly isolated strain, Desulfovibrio desulfuricans strain CSN, was used throughout this study. This strain has a very versatile energy metabolism and has already been used in a study on disproportionation of sulfur compounds

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, dicyclohexylcarbodiimide

Materials and methods

Organism

238 (Cypionka and Krfimer 1989). Strain CSN was isolated from anoxic sludge of a ditch near Konstanz after two weeks of enrichment in a chemostat culture containing freshwater medium with lactate (30 mM) and limiting concentrations of sulfate (5 mM) plus nitrate (3 raM). A pure culture was obtained from repeated deep agar dilution series (Widdel and Pfennig 1984). The cells were Gram-negative, motile, slightly curved rods. Strain CSN was able to use Hz, formate, ethanol, lactate, or succinate as electron donor with sulfate, thiosulfate, dithionite, elemental sulfur, nitrate, or nitrite as electron acceptor. Nitrate and nitrite were reduced to ammonia. Pyruvate, fumarate, or malate could be fermented in the absence of inorganic electron acceptors. Acetate, methanol, propionate, butyrate, benzoate, trimethoxybenzoate, glucose and fructose were not utilized as energy substrates. For growth with H2 + CO2 or formate, an additional carbon source such as acetate was required. In the absence of electron donors, sulfite or thiosulfate were disproportionated to sulfate and sulfide (Bak and Cypionka 1987; Bak and Pfennig 1987); however, growth was observed only with sulfite, but not with thiosulfate as the only energy source. Desulfoviridin and cytochromes (c- and b-type) were present. The G + C content of the D N A was determined as 59.45 _+ 0.25 tool% by the thermic denaturation method (Marmur 1961; DeLey 1970). On the basis of morphology, the substrates utilized, the G + C content of the DNA, and the ability to reduce nitrate and nitrite, strain CSN was classified as Desulfovibrio desulfuricans.

Cultivation Desulfovibrio desulfuricans was grown with H2 and limiting concentrations of sulfate in sulfide- and pH-controlled chemostat culture as previously described (Cypionka 1986, 1987; Seitz and Cypionka 1986, Cypionka and Pfennig 1986). The defined mineral medium contained 2 m M acetate as additional carbon source.

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Fig. 1. Uptake experiment with Desulfovibrio desulfuricans. Washed

cells (4 mg protein) were incubated at - t ~ in 4 ml Ha-saturated 150 mM KC1. pH ( - - - - ) and the (logarithmic) signal of the sulfide electrode (...... ) were recorded by means of a microcomputer. The sulfide concentration (+) was online calculated from the sulfide electrode signal after correction for pH-changes. Additions (in nmol) are indicated at the arrows

of a non-centrifuged sample of the cell suspension. If only a small fraction of the added sulfate had been taken up by the cells, the balance (recovery in the supernatant plus recovery in the cells compared with radioactivity of the suspension) was nearly 100%. If most of the sulfate had been taken up, the recovery was lower (87 _+ 10%), as some loss occurred during centrifugation and resuspension of the cells. Therefore, uptake was normally calculated from the radioactivity removed from the supernatant. A standard experiment as described above was performed as a control at every experimental day, and all results were calculated by comparison with the standard experiment of the same day. All experiments were carried out at least two times at different days.

Uptake experiments with labelled sulfate Uptake experiments were performed with washed cells in a 4 ml glass vessel with Hz-saturated 150 m M KC1 containing 1 ~tg of the redox indicator resazurin. Proton movements and sulfide formation were followed by means of electrodes (Cypionka 1987). Data were recorded by an Apple II + computer using a 12 bit analog/digital converter (Model 1208 A, Technosystems, Darmstadt, FRG). This allowed online calculation of the sulfide concentration from the logarithmic signal of the sulfide electrode, and simple correction for the pH sensitivity of the sulfide electrode. Cells were added to a concentration of I mg protein (3.6" 10 9 cells) per ml and allowed to equilibrate for 30 min at room temperature. Then, the assay was cooled to - 1.2 ~C to prevent immediate sulfide formation from added sulfate. To start the uptake experiment, usually 5 or 10 nmol (1.25 or 2.5 IxM) of 35S-labelled sulfate (50 Bq/nmol) was injected by means of a Hamilton syringe. After 2.5 min, a sample (1 ml) was withdrawn, and the cells were centrifuged for I rain through 0.2 ml silicone oil (Serva D C 704, d = 1.07) that was adjusted to a buoyant density of 1.035 with nhexadecane ( d = 0.77). The radioactivity of the cell-free supernatant and of the cells (resuspended in 0.5 ml KC1 after removal of the silicone oil) was counted in Lumagel (Baker, Deventer, The Netherlands), and was compared with that

Results

Sulfate uptake by Desulfovibrio desulfuricans in H2-saturated KCl at - 1~C A typical uptake experiment at - 1 ~ is shown in Fig. 1. After calibration pulses of 5 nmol sulfide and 10 nmol H +, the addition of 5 nmol (1.25 ~tM) of 35S-labelled sulfate to a cell suspension in H2-saturated 150 m M KC1 resulted in an alkalinization of the medium without immediate formation of sulfide. About 2 nmol of H + disappeared per nmol sulfate (in some experiments less proton uptake was observed). Analysis of the sample taken after 3 rain revealed that 91% of the sulfate added had been removed from the supernatant. This corresponds to 1.15 nmol SO2-/mg protein or 2.105 sulfate molecules per cell. Assuming a cytoplasmic cell volume of 1.4 lal/mg dry mass (Varma et al. 1983) the intracellular sulfate concentration would amount to 0.64 mM, whereas the remaining concentration in the supernatant was only 0.11 pM. Thus, the cells accumulated suIfate 5,820-fold, This accumulation factor has, however, to be judged with some caution as it changes drastically with slight changes of the amount taken up by the cells. I f sulfate uptake varies by the standard deviation of 4_=-6% (42 experiments), the resulting accumulation factors are 26,650 and 3,340.

239 Table l. Influence of experimental and growth conditions on sulfate accumulation by Desulfovibrio desulfuricans Conditions

Percent uptake of control with sulfate-limited cells in H2-saturated 150 mM KC1 at - I ~ and pH 7, with the addition of 2.5 gM sulfate

Sample taken after reduction of sulfate at room temperature

26

Cells suspended in 30 mM KC1

104

Cells suspended in 150 mM NaC1

107

Addition of 0.2% isopropanol or dimethyl sutfoxide

100

Cells incubated under air for 10 min for 120 min

100 50

Cells pasteurized for 10 min at 80~C pH 5.5 pH 8.5

1 103 12 to 75

Cells grown with excess sulfate

74 to 100

Cells grown with thiosulfate

51

Influence of experimental and growth conditions The low temperature ( - 1~C) during the uptake experiments was required to prevent immediate sulfide production from the sulfate added. Only a quarter of the label was associated with the cells if the cells were incubated at room temperature and the sample was withdrawn 3 rain after reduction of the added sulfate (Table 1). Obviously, most of the label had left the cells as HzS after this time. No differences in sulfate accumulation were observed if the cells were washed and suspended in 30 mM KC1 or in 150 mM NaC1, or when 0.2% isopropanol or dimethyl sulfoxide, that were used for dissolving some of the inhibitots tested, were added. Although sulfate-reducing bacteria are strictly anaerobic, oxygen did not immediately inhibit sulfate accumulation. Sulfate accumulation was not diminished after 10 rain incubation under air. After 2 h of incubation under air, however, sulfate uptake was inhibited by 50%. Pasteurized cells did not accumulate sulfate. At pH 5.5, sulfate accumulation and reduction were not affected. At pH 8.5, sulfate reduction was completely inhibited, and sulfate uptake was variably inhibited by 25 to 88%. This inhibition was completely reversed by changing the pH to 7 or 5.5. Cells of Desulfovibrio desulfuricans grown with excess sulfate accumulated 74 to 100% of the amount taken up by sulfate-limited cells. Cells grown with thiosulfate as electron acceptor took up 51% compared with sulfate-limited cells (Table 1).

Kinetics and dependence on the sulfate concentration The experimental procedure used throughout this study did not allow the measurement of fast uptake kinetics. Usually, the sample was withdrawn after 2.5 min and centrifuged

within the next minute. Thus, the results give the state 3 min after sulfate addition. If 1.25 or 2.5 gM sulfate had been added, a steady state was reached at this time and the accumulation did not further increase with time (Table 2). After addition of 12.5 gM sulfate, the same accumulation factor as before was reached after 6 min. The intracellular sulfate concentration increased proportional to the added sulfate concentration. With further increasing sulfate concentrations (0.125 to 1.25mM) the accumulation factor drastically decreased down to 8. The intracellular sulfate concentration did not exceed 25.6 mM. At - 1 ~C, the maximum uptake rate was 12 nmol per min per mg protein after the addition of 1.25 mM sulfate. In experiments carried out in Nz-saturated KC1 at 20 ~C, a rate of 36.6 nmol per min per mg protein was determined for the first minute. However, at this time the steady state concentration was already reached indicating a higher maximum uptake rate.

Reversibility Sulfate transport was reversible. Accumulated sulfate was rapidly lost from the cells by washing with 1 mM sulfate (in 150 mM KC1), whereas it was not released by washing with sulfate-free KC1. If 3 min after addition of 2.5 gM labelled sulfate a 100-fold excess of non-labelled sulfate was added, after further 3 rain about 80% of the label was detected in the snpernatant (Table 2). Most of the label could be precipitated with BaCI2 indicating that it was still existing as sulfate. The accumulation after 6 rain corresponded to that expected for an initial addition of 250 ~tM sulfate. Thus, an equilibrium between labelled and non-labelled sulfate was adjusted within 3 min at - 1 ~C. This indicated rapid exchange between intracellular and extracellular sulfate. Another method to release accumulated sulfate was the addition of the uncoupler CCCP (see below).

Influence of ATPase inhibitors The addition of DCCD (100 nmol/mg protein) resulted in a short acidification followed by a drift to more alkaline pH for about 15 rain. Sulfite and thiosulfate were still reduced in the presence of DCCD, whereas sulfate reduction was inhibited. Uptake of sulfate, however, was not affected, although there was no significant alkalinization of the suspension medium during sulfate uptake (Fig. 2, Table 3). Oligomycin ( 5 0 - 1 0 0 nmol/mg protein) did not inhibit sulfate reduction and was not added to sulfate transport experiments. The phosphate analogue arsenate did not affect sulfate reduction and uptake at a concentration of 5 mM.

Influence of ionophores After 30 min preincubation with the uncoupler CCCP, sulfate reduction was irreversibly inhibited (Table 3). The addition of sulfate did not cause alkalinization and sulfate uptake was almost completely inhibited. The addition of CCCP 2 rain after sulfate caused release of more than 50% of the accumulated sulfate within 3 min. The channel former gramicidin S ( 1 0 - 50 nmol/mg protein) caused no significant inhibition of sulfate reduction. The cells remained motile and were not deenergized. Uptake

240 Table 2 Influence of the sulfate concentration on sulfate accumulation at - 1~C

Sulfate added (l,tM)

Sample after (min)

Uptake (nmol/mg protein)

Intracellular concentration ~ (mM)

Residual conc. (pM)

Accumulation

1.25

3 6

1.18 1.17

0.64 O.63

0.11 0.12

5820 5250

2.50

3 6

2.28 2.28

1.24 1.24

0.22 0.22

5640 5640

With the addition of 125 gM non-labelled sulfate after 3 min: 6 12.5 a Assuming 1.4 gl cytoplasmic volume per mg cell dry mass (Varma et al. 1983) corresponding to 1.84 gl per mg protein b Experiment carried out at 20~C in N2-saturated KC1

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118

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8.5 11.5

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Fig. 2. Influence of dicyclohexylcarbodiimide (DCCD) on sulfate reduction and uptake by Desulfovibrio desulfurieans. Washed cells (1.2 mg protein) were incubated at 30~C in 4 ml Hz-satured 150 mM KC1 with 100 gM DCCD. Additions (in nmol) are indicated at the arrows. Note that sulfate was not reduced, and that the addition of sulfate did not cause significant alkalinization. The labelled sulfate, however, was almost completely taken up and accumulated 6,200fold

111 100

74 141

19.6 25.6

1215 1205

16 21

19.9

2464

8

N a + - H + exchange (Krulwich 1983), did not inhibit sulfate accumulation and reduction. The addition of the K + transporter valinomycin caused acidification of the suspension medium followed by proton backflow within further 3 min. Valinomycin did not inhibit sulfate reduction or sulfate accumulation. Even the combination of valinomycin plus nigericin, added to dissipate A 7j and ApH, did not affect sulfate accumulation. Obviously, valinomycin did not dissipate A ~ in D. desulfuricans. Cells incubated in 150 m M K S C N (in order to destroy A ~ by the permeant thiocyanate anion) were able to reduce sulfite or thiosulfate. Sulfate was only reduced if it was added in higher concentration (1 mM). In the presence of thiocyanate sulfate uptake was inhibited by 60%. The addition o f valinomycin did not enhance inhibition, whereas with additional nigericin only 26% were taken up. The p H dependence of sulfate uptake was also studied in the presence o f KSCN. At pH 8.5, sulfate accumulation was inhibited almost completey. After changing the p H to 7 the uptake increased again to 39% (Table 3).

Influence of structural analogues experiments in the presence of gramicidin were not performed. The addition o f the K +-H + antiporter nigericin resulted in a slow alkalinization of the suspension medium indicative for proton uptake during dissipation of ApH. When testing nigericin as inhibitor, variable results were obtained. In some experiments the reduction of micromolar sulfate additions was inhibited, but could be stimulated by the addition o f sulfite or thiosulfate. Higher concentrations of sulfate (1 mM) could always be reduced. The cells were not deenergized and remained motile. In transport experiments 60 to 100% compared with the control experiment were taken up. The N a + - H + antiporter monensin was added to cell suspensions incubated in 150 m M NaC1. Under these conditions, sulfate reduction was not inhibited and the cells took up 82% o f the control experiment. Amiloride, an inhibitor o f

The addition o f 0.1 m M molybdate completely inhibited sulfate reduction, whereas reduction of sulfite or thiosulfate was not affected. Accumulation of sulfate, however, was not diminished in the presence o f molybdate (Table 4). Tungstate (0.1 mM) did not affect uptake and reduction of sulfate, whereas 0.1 m M chromate caused complete inhibition o f these activities. Selenate additions (1.25 or 2.5 pM) caused the same alkalinization as observed with sulfate. At r o o m temperature, selenate resulted in a heavy increase of the signal o f the silver/silver sulfide electrode. This was obviously due to the formation of selenide that reacted with the sulfide electrode. (The solubility product o f silver selenide, 1.6.10-5 s, is even lower than that of silver sulfide, 6.6" 10-s~ The reduction of selenate appeared not to be complete and stopped after several pulses. These results correspond to those recently reported by Zehr and Oremland (1987).

241 Table 3. Influence of DCCD, arsenate and ionophores on sulfate accumulation a Inhibitor

Concentration

Percentuptake of control

DCCD b (30 min preincubation)

100 gM

102

5 mM

107

CCCP if added 2 rain after 3sSO4a-

50 gM

3 46

Valinomycin

50 gM

105

Nigericin

50 gM

Arsenate

Nigericin + valinomycin

60 to 100 104

Monensin (in 150 mM NaC1) Amiloride (in KC1 or NaC1) KSCN

100 gM

82

2 mM

103

150 mM

39

KSCN + valinomycin

46

KSCN + valinomycin + nigerlcin

26

KSCN at pH 8.5 2 min after pH change to 5.5

9 39

" Experiments were performed at - 1 ~ after 1 h preincubation with the inhibitors at room temperature. Cell concentration was i nag protein/ml and sulfate added 2.5 gM b Similar results were obtained at - t and at 30~ (see Fig. 2)

Table 4. Influence of structural analogues on sulfate uptake by

Desulfovibrio desulfuricans Analogue a

Percent uptake of control

Molybdate Tungstate Chromate Selenate Thiosulfate Sulfite Selenite

105 107 2 50 39 85 107

a The analogues were added to a concentration of 100 I~M 5 min prior to 2.5 rtM of labelled sulfate at - 1 ~

by oxygen. This result differs from that reported by Furusaka (1961), however, it corresponds with the observation that in the absence of sulfide oxygen is not immediately toxic for sulfate-reducing bacteria (Cypionka et al. 1985). The kinetics of sulfate transport could not be characterized very accurately in this study; however, the maximum rates were in the range required for the growth rates observed in chemostat culture. The uptake rates, but not the steady state accumulation, were temperature-dependent.

Absence of an ATP-dependent transport mechanism The sulfate molecule has to be activated with ATP prior to reduction. At first sight, an ATP-driven sulfate transport system immediately activating sulfate to APS (comparable to the phosphotransferase system in enterobacteria) would represent a very elegant solution of sulfate uptake. However, inspire of ATP consumption the ATP sulfurylase reaction has an unfavourable free energy change ( + 46 kJ/mol under standard conditions at pH 7), and appears not to be appropriate to drive sulfate transport additionally. If 1 ATP was consumed only for moving sulfate across the membrane the cell would have to hydrolyze 3 phosphate ester bonds (including the pyrophosphate formed by ATP sulfurylase) for transport and activation before sulfate could be reduced within the ceil. Many assimilatory bacterial sulfate transport systems are ATP-dependent and involve a periplasmic binding protein (Dreyfuss 1964; Pardee et al. 1966; Furlong 1987; Ames 1988). Periplasmic transport systems are sensitive to arsenate and DCCD. With Desulfovibrio desulfuricans, arsenate did not affect sulfate transport or sulfate reduction. The cells appeared to be resistant to this phosphate analogue. DCCD inhibited sulfate reduction, while sulfate tranport was not affected. Thus, DCCD might have caused removal of intracellular ATP required for sulfate activation, while ATP appeared not to be involved in sulfate transport.

Electrogenic mechanism

General characteristics of sulfate transport

The membrane potential, ATP level, and the gradients of H+, K+, Na + across the cytoplasmic membrane of Desulfovibrio desulfuricans have not been studied so far. Therefore, it is not assured whether the various ionophores exerted the effects they are supposed to. Strain CSN appeared to be quite resistant to some inhibitors as was already observed with D. desulfuricans strain Essex (Cypionka 1987). However, several independent results favour the view that sulfate is taken up electrogenically in symport with protons: (i) Sulfate transport was reversible, and after addition of an excess of non-labelled sulfate, most of the label was rapidly lost from the cells. Reversibility implies that sulfate accumulation is in equilibrium with Afin +. Under these conditions it appears very unlikely that an accumulation factor of 5,000 should be sustained by Aptt as the only driving force. The steady state accumulation of an anion reversibly transported in symport with protons is described by

Desulfovibrio desulfuricans strain CSN is able to highly ac-

log (ci/Co)

cumulate micromolar additions of labelled sulfate as recently described for the Essex strain of the same species (Cypionka 1987). Although sulfate-reducing bacteria are strict anaerobes, accumulation was not immediately affected

with ci, Co= concentrations inside and outside the cell, m = charge of the anion, n = number of symported protons, A 7~ = electrical potential (mV), Z = 2.3-R.T/F ~ 60 mV, ApH = pH gradient (pH~ -- pHo).

Although selenate did not inhibit sulfate reduction the uptake of sulfate was significantly diminished. Significant inhibition of sulfate accumulation was also obtained with thiosulfate, whereas sulfite inhibited sulfate uptake only slightly. The addition of selenite caused disappearance of sulfide present in the suspension, probably by oxidation to elemental sulfur. Uptake and reduction of sulfate were not inhibited by 0.1 mM selenite (Table 4).

Diseussion

=

--

(m + n) A ~P/Z + n" A p H ,

242 Assuming typical values of 0.5 for ApH and - 1 5 0 mV for A ~, electroneutral symport with 2 H + would result in a tenfold accumulation, whereas electrogenic symport with 3 H + would allow 10,000-fold sulfate accumulation. (ii) The inhibitors added to dissipate ApH (nigericin in KC1, monensin in NaC1) never completely inhibited sulfate accumulation. While at an external pH of 8.5 sulfate reduction was completely inhibited and ApH (inside alkaline) should be dissipated or even inversed, there was still considerable accumulation. Acid milieu did not affect sulfate accumulation and reduction. These results indicated that the pH gradient is not the only driving force of sulfate accumulation. (iii) The uncoupler CCCP, that completely deenergizes the cells after prolonged incubation, is assumend to dissipate A ~ (that has a low capacity compared with ApH) prior to ApH. The rapid loss of accumulated sulfate upon addition of CCCP indicated the involvement of A 7j in sulfate accumulation. (iv) The cells turned out to be insensitive to valinomycin which is often used to dissipate A ~v. The dependence of sulfate accumulation on A ~f (and ApH) could, however, be demonstrated in the presence of the permeable thiocyanate anion. Sulfate uptake (probably driven by ApH) was diminished at pH 7 and completely (but reversibly) inhibited at alkaline pH. (v) There were no indications for an involvement of Na + or K +. It made no difference whether transport experiments were carried out in NaC1 or in KC1. Amiloride, an inhibitor of Na +-H + exchange was without influence. (vi) Proton uptake during sulfate accumulation as observed with a pH electrode was not always correlated with the amount of sulfate taken up. In several experiments, there was high sulfate accumulation without any significant alkalinization of the suspension medium. Thus, the stoichiometry of H +-sulfate symport could not be determined by means of a pH electrode.

Specificity of sulfate transport Selenate and thiosulfate are classical inhibitors of assimilatory sulfate transport in various organisms (Cuhel et al. 1981; Utkilen et al. 1976; Jeanjean and Broda 1977; Yamamoto and Segel 1966; McCready and Din 1974; Tweedie and Segel 1970; Vange et al. 1974; Dean 1975). These anions diminished sulfate accumulation also in D. desuIfuricans. As only the steady state accumulation but not kinetics of sulfate transport have been studied there was no indication whether selenate and thiosulfate act as competitive inhibitors (Postgate 1949). Obviously, selenate, thiosulfate and sulfite could be taken up and reduced. Accumulation of these anions could possibly interfere with the driving forces for sulfate transport. Molybdate, although specifically inhibiting sulfate reduction, did not affect sulfate accumulation. This fits with the reports that molybdate causes ATP depletion by reacting with ATP sulfurylase to give an unstable product that decays to molybdate and AMP (Wilson and Bandurski 1958; Taylor and Oremland 1979). The strong inhibitory effect of chromate might be unspecific as chromate is a strong oxidizing agent that immediately oxidized the transport assays. Insensitivity towards tungstate, molybdate and selenite indicated high specificity compared with most assimilatory sulfate transport systems.

Energy requirement As the free energy change of dissimilatory sulfate-reduction is low, the energy requirement for sulfate transport has to be considered. In a previous study (Cypionka 1987) it was discussed that an electroneutral uptake mechanism (symport of sulfate with 2 H +) would not require net energy. If the membrane-bound ATPase required 3 H + per ATP, and 2 H + finally left the cell with H2S, the net energy requirement of sulfate uptake with 3 H + and end-product release would amount to 1/3 ATP. High accumulation factors were observed only after micromolar sulfate additions. With increasing sulfate concentrations the accumulation factor decreased significantly. So far it is not known how sulfate uptake regulated. The presence of different transport systems is conceivable. The spore-forming sulfate reducer Desulfotomaculum orientis accumulated sulfate only if grown under sulfate-limiting conditions (unpublished work). Growth yields with H2 plus sulfate obtained in chemostat culture were significantly (by 22% or about 2 g dry mass per mol sulfate) higher under hydrogen-limiting than under sulfate-limiting conditions (Cypionka and Pfennig 1986). Probably this strain has two different sulfate uptake systems with different energy requirement.

Acknowledgements. The author thanks Prof. R. K. Thauer for valuable advice and Prof. N. Pfennig for his stimulating support. Waltraud Dilling determined the G + C content of the DNA. This work was supported by the Deutsche Forschungsgemeinschaft. References Ames GF-L (1988) Structure and mechanism of bacterial periplasmic transport systems. J Bioenerget Biomembr 20:1 - 18 Bak F, Cypionka H (1987) A novel type of energy metabolism involving fermentation of inorganic sulphur compounds. Nature 326: 891 - 892 Bak F, Pfennig N (1987) Chemolithotrophic growth of Desulfovibrio sulfodismutans sp. nov. by disproportionation of inorganic sulfur compounds. Arch Microbiol 147:184 - 189 Cuhel RL, Taylor CD, Jannasch HW (1981) Assimilatory sulfur metabolism in marine microorganisms: Characteristics and regulation of sulfate transport in Pseudomonas halodurans and Alteromonas luteo-violaceus. J Bacteriol 147: 340- 349 Cypionka H (1986) Sulfide-controlled continuous culture of sulfatereducing bacteria. J Microbiol Meth 5 : 1 - 9 Cypionka H (1987) Uptake of sulfate, sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfurieans. Arch Microbiol 148 : 144-149 Cypionka H, Pfennig N (1986) Growth yields of Desulfotomaeulum orientis with hydrogen in chemostat culture. Arch Microbiol 143 : 396- 399 Cypionka H, Widdel F, Pfennig N (1985) Survival of sulfate-reducing bacteria after oxygen stress, and growth in sulfate-free oxygen-sulfide gradients. FEMS Microbiol Eco131:39-45 Deane EM (1975) Sulphate uptake and metabolism in the chrysomonad Monochrysis lutheri. Arch Microbiol 105:295- 301 DeLey J (1970) Reexamination of the association between melting point, buoyant density and the chemical base composition of deoxyribonucleic acid. J Bacteriol 101:738--754 Dreyfuss J (1964) Characterization of a sulphate and thiosulphate transporting system in Salmonella typhimurium. J Biol Chem 239:2292-2297 Furlong CE (1987) Osmotic-shock-sensitive transport systems. In: Neidhardt FF (ed) Eseherichia coli and Salmonella typhimurium: cellular and molecular biology, ASM, Washington DC, pp 768-- 796

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Received December 23, 1988/Accepted April 20, 1989