Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK1
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK2
Author for correspondence: Ben C. Berks (at Oxford address). Tel: +44 1865 275250. Fax: +44 1865 275259. e-mail: ben.berks{at}bioch.ox.ac.uk
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ABSTRACT |
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Keywords: tetrathionate reductase, thiosulfate reductase, polysulfide reductase, molybdopterin, electron transport
Abbreviations: MGD, bis(molybdopterin guanine dinucleotide)molybdenum
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INTRODUCTION |
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The use of a molybdopterin cofactor to catalyse these reactions is intriguing since this cofactor normally catalyses oxygen atom transfer, hydrogen atom transfer or a combination of the two (Hille, 1996 ; Khangulov et al., 1998
). The enzymes carrying out sulfur bond cleavage (which might alternatively be styled sulfur atom transfer) therefore operate a previously undescribed biological reaction of the molybdopterin cofactor. Sequence similarities between the MGD-binding subunits of Ttr, Phs and Psr in the polypeptide region that contributes to the enzyme active-site pocket and substrate-access channel support the idea that these proteins form a specificity grouping distinct from other MGD-dependent enzymes (Hensel et al., 1999
; Fig. 1
).
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METHODS |
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For anaerobic growth on solid media, plates were incubated in gas jars under a nitrogen/CO2 atmosphere generated using the Anaerogen system (Oxoid). Tests for growth on the non-fermentable carbon source glycerol utilized the minimal medium described by Pope & Cole (1982) but lacking the nutrient broth component and supplemented with 30 mM glycerol and the electron acceptor of interest. Dispersed elemental sulfur was introduced into solid media by air oxidation of plates prepared with a polysulfide solution as described by Moser & Nealson (1996)
.
Genetic techniques.
Standard molecular genetic techniques were carried out as described by Sambrook et al. (1989) . P22-mediated transductions were performed as detailed in Maloy et al. (1996)
using P22 HT int. Point mutations at the Cys256 TGT codon in ttrA were constructed using the Quick Change System (Stratagene). The new codons, GCT for the Ala mutation and TCT for the Ser mutation, were first introduced into the XbaI fragment of ttrA that bears codon 256 and then used to replace the corresponding XbaI wild-type fragment in pAH26.
Analytical methods.
Trithionate was synthesized as described by Kelly & Wood (1994) . Tetrathionate, trithionate and thiosulfate concentrations were determined by cyanolysis according to the protocols of Kelly & Wood (1994)
. Assays for methyl-viologen-linked activities and for polysulfide reductase activity were carried out in glass cuvettes sealed with a butyl rubber stopper and rendered anoxic by bubbling with oxygen-free nitrogen. For the methyl-viologen-linked measurements the assay solution contained 10 mM sodium phosphate buffer pH 7·4, 2 mM Na2EDTA and 1 mM methyl viologen. The methyl viologen was reduced by titration with a Ti(III) citrate solution until the A600 reached 1·5. The reaction was started by the addition of electron acceptor, and oxidation of methyl viologen radical was monitored at 600 nm [
600 (MV·+-MV2+)=13 mM-1 cm-1]. Electron acceptors were used at the following concentrations: 500 µM potassium tetrathionate; 500 µM potassium trithionate; 1 mM sodium thiosulfate. Tetrathionate and trithionate reductase assays had to be corrected for the rate of chemical reduction of the substrate by the methyl viologen radical. For measurement of polysulfide reductase activity the assay buffer was 50 mM Tris/HCl, pH 8·3. Polysulfide was added to a concentration of 1 mM from a stock solution prepared from sodium sulfide and sulfur flowers as described by Moser & Nealson (1996)
. Bacteria were introduced into the cuvette, following which the assay was initiated by addition of 10 mM sodium formate to act as electron donor to the bacterial electron-transfer chain. Reduction of polysulfide was monitored via the decrease in A360 using
=0·38 mM-1 cm-1 (Klimmeck et al., 1998
).
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RESULTS AND DISCUSSION |
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Phs does not contribute to physiological tetrathionate oxidation (Hensel et al., 1999 ). However, it is not clear that Phs is expressed while tetrathionate is present in the growth medium. It was therefore possible that Phs lacks physiological tetrathionate reductase activity not for mechanistic reasons but because it is not expressed under the appropriate growth conditions. This idea was tested by measuring tetrathionate reductase activity in a ttr mutant that had been grown under conditions suitable for the induction of Phs synthesis. No significant tetrathionate reductase activity could be measured in these cells with either MV·+ or physiological electron donors even though thiosulfate reductase activity was easily detected. This experiment suggests that Phs is not mechanistically capable of reducing tetrathionate.
Ttr and Phs are capable of oxygen atom transfer
It has previously been reported that Phs is capable of reducing chlorate to chlorite (Riggs et al., 1987 ). This is a significant observation because the reduction of chlorate involves an oxygen atom transfer rather than the sulfursulfur bond cleavage of the physiological thiosulfate reduction reaction. To test whether Ttr is also capable of oxygen atom transfer we cultured phs and phs ttr mutants in the presence of tetrathionate to induce Ttr expression, and then measured MV·+-linked chlorate reductase activity in whole cells. A specific chlorate reductase activity of 190 nmol MV·+ oxidized min-1 (mg protein)-1 was measured for the strain APH8 (ttr+ phs asr) compared to 70 nmol MV·+ oxidized min-1 (mg protein)-1 for strain APH12 (ttr phs asr). Thus the Ttr+ strain had a chlorate reductase activity almost three times higher than that of the Ttr- strain. The S. enterica membrane-bound nitrate reductase is also known to have chlorate reductase activity (Barrett & Riggs, 1982
). Control measurements showed that both the Ttr+ and Ttr- strains had low, but identical, nitrate reductase activities [29 nmol MV·+ oxidized min-1 (mg protein)-1], indicating that the difference in chlorate reductase activity between the strains cannot be ascribed to nitrate reductase. This experiment suggests that Ttr has chlorate reductase activity and that the Ttr molybdopterin cofactor is capable of oxygen atom transfer.
Azide is a specific inhibitor of some molybdopterin-dependent enzymes (e.g. membrane-bound nitrate reductases: Craske & Ferguson, 1986 ; E. coli formate dehydrogenase-H: Axley et al., 1990
). However, azide at a concentration of 100 µmol l-1 did not inhibit the tetrathionate reductase activity of Ttr or the thiosulfate reductase activity of Phs.
Reduction of trithionate by S. enterica
It was of interest to determine whether the Ttr enzyme specifically reduces tetrathionate or whether it is also able to cleave polythionate compounds with other chain lengths. Trithionate () can be formed by the facile chemical reaction of tetrathionate and sulfite (reaction 4) and is therefore likely to be present in many tetrathionate-containing environments.
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We found that S. enterica is capable of anaerobic growth on the non-fermentable carbon source glycerol with trithionate as sole respiratory electron acceptor. This growth was dependent on the Ttr enzyme since ttr mutants failed to grow under these culture conditions. In contrast, trithionate-linked growth was unaffected in a phs mutant. E. coli strain MC4100 was found to be unable to utilize trithionate as terminal electron acceptor. However, introduction of plasmid pAH26 carrying the ttrRSBCA cluster allowed growth of this strain on trithionate, confirming that Ttr is responsible for trithionate-dependent growth.
The products of the trithionate reduction reaction were examined in wild-type S. enterica LT2a during growth on minimal glycerol medium. For each mole of trithionate consumed approximately one mole of thiosulfate and one mole of sulfite were produced. This suggests that trithionate is reduced by Ttr in a reaction (equation 5) analogous to that of tetrathionate reduction.
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The ability of Ttr and of Phs to reduce trithionate was also tested using the non-physiological electron donor MV·+. Cells cultured in the presence of trithionate exhibited similar rates of MV·+-linked trithionate and tetrathionate reduction. This trithionate reductase activity was absent from a ttr but not a phs strain, demonstrating that trithionate reduction requires Ttr (Fig. 2). To be sure that Phs does not have trithionate reductase activity the ttr mutant was cultured in the presence of thiosulfate rather than trithionate to ensure high Phs expression. Even under these conditions of Phs induction no significant methyl-viologen-dependent trithionate reduction could be measured.
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Expression of the ttrBCA operon requires the TtrSR two-component regulator and is induced by tetrathionate (Hensel et al., 1999 ; Price-Carter et al., 2001
). It is probable that tetrathionate is sensed directly by TtrS since thiosulfate, the product of tetrathionate metabolism, does not induce Ttr expression. In this study we found that Ttr was induced in medium lacking tetrathionate but containing trithionate. This suggests that trithionate is capable of inducing Ttr synthesis and therefore that TtrS may be capable of sensing both trithionate and tetrathionate.
Reduction of elemental sulfur by S. enterica
We investigated whether the sulfursulfur bonds of elemental sulfur were also amenable to cleavage by S. enterica. Solid medium containing dispersed solid sulfur was prepared by air oxidation of agar plates to which an alkaline polysulfide solution had been added at the time of pouring.
S. enterica LT2a was capable of anaerobic growth on LB agar plates containing 50 mM formate and 10 mM elemental sulfur. The opaque elemental sulfur precipitate within the plates cleared around the colonies, indicating that the insoluble sulfur had been converted to another species by bacterial metabolism (Fig. 3a). In the absence of formate, which acts as a direct electron donor to the respiratory electron transfer chain, sulfur clearing still occurred but was not as extensive. Sulfur clearing was also observed on solid minimal medium containing 50 mM formate with 0·4% lactate as carbon source. E. coli, which does not utilize inorganic sulfur compounds as respiratory electron acceptors, did not exhibit the sulfur-clearing phenotype (Fig. 3a
). Sulfide (up to 5 mM) could be detected in cultures of S. enterica growing anaerobically in liquid LB medium in the presence of sulfur powder, suggesting that sulfide is the product of the sulfur dissolution reaction. Significant quantities of tetrathionate or thiosulfate were not detected in these liquid cultures.
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Elemental sulfur did not support growth of S. enterica in the presence of a non-fermentable carbon source. This observation is congruent with the involvement of Psr in sulfur reduction since S. enterica cannot grow by thiosulfate respiration alone. Instead thiosulfate reduction functions to augment growth on fermentable carbon sources (Heinzinger et al., 1995 ; A. P. Hinsley & B. C. Berks unpublished).
Sulfur clearing by S. enterica was prevented by addition of 30 mM tetrathionate to the plates. This tetrathionate inhibition was relieved in a ttr mutant, indicating that tetrathionate must be metabolized to suppress sulfur clearing. Addition of 20 mM thiosulfate to the sulfur plates did not inhibit sulfur clearing. This shows that it is not the product of the Ttr reaction that is responsible for suppression of sulfur clearing. Instead the effect of tetrathionate is most likely due to the preferential routeing of reducing equivalents to Ttr rather than Phs, a hierarchy previously observed in the differential utilization of tetrathionate and thiosulfate (e.g. Hensel et al., 1999).
Mechanisms for the sulfur-clearing phenotype in which the cells directly reduce elemental sulfur can be ruled out since (i) the sulfur clearing extends far beyond direct contact with the cells (Fig. 3a), (ii) sulfide production from sulfur can still be measured in liquid culture when either the cells or the sulfur powder are placed inside a sealed dialysis sac and (iii) the thiosulfate reductase active site is in the periplasmic compartment, which the insoluble sulfur substrate cannot access. Two possible models are suggested for the metabolism of sulfur by S. enterica using water-soluble species: thiosulphate cycling and polysulfide reduction.
The thiosulfate cycling model (Fig. 4a) assumes that trace quantities of thiosulfate are present in the sulfur plates. This is not unreasonable since the stock polysulfide solution has been oxidized by molecular oxygen. The thiosulfate is metabolized by the normal Phs reaction (overall equation 6). The product sulfite molecule, produced at the periplasmic face of the inner membrane, diffuses away from the cell and reacts by well-established and facile chemistry with elemental sulfur producing thiosulfate (equation 7). Since reaction (7) produces one mole of thiosulfate for each mole of thiosulfate reduced in equation (6) the combination of the two reactions results in the reduction of elemental sulfur to sulfide, with thiosulfate having a catalytic role in the process (equation 8). Possible support for the thiosulfate cycling mechanism comes from the observation that strains that are capable of thiosulfate reduction but are unable to reduce the sulfite product (the S. enterica asr mutant and E. coli expressing phs) reproducibly show larger zones of sulfur clearing than strains that can reduce both thiosulfate and sulfite (Fig. 3a
).
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The polysulfide reduction mechanism requires that Phs can operate as a polysulfide reductase. However, we found that washed cells of S. enterica that had been induced for Phs expression did not reduce polysulfide with formate as electron donor. Similarly no polysulfide reductase activity could be detected with cells induced for Ttr expression. A caveat with these measurements is that the polysulfide assay is relatively insensitive and might not pick up low-level polysulfide reductase activity [of the order of 10 nmol polysulfide sulfur reduced min-1 (mg protein)-1 or less] associated with Phs. Nevertheless, our polysulfide reductase activity measurements provide no positive support for the idea that a polysulfide reduction mechanism is operating during sulfur clearing by S. enterica. It is also worth noting that addition of 20 mM thiosulfate to the plates did not inhibit the sulfur-clearing reaction (above). Since thiosulfate is a known substrate of Phs, such a high concentration of thiosulfate would be expected to competitively inhibit any polysulfide reductase activity of the enzyme if it existed.
Polysulfides are unstable at low pH, disproportionating to elemental sulfur and sulfide according to equation (9). We considered that an analysis of the pH dependence of the sulfur-clearing phenotype catalysed by S. enterica might give insight into the possible involvement of polysulfides in the process. The ability of S. enterica to clear sulfur was tested on LB-formate-sulfur plates buffered by means of a 50 mM concentration of an appropriate Good buffer. A range of pH values in the interval 6·09·0 was examined. While sulfur clearing was most efficient at high pH the phenotype was still detectable at pH 6·0 (Fig. 3b). The solubility of polysulfide in a 10 mM solution of sulfide at pH 6·0 and 37 °C is approximately 10 µM (Schauder & Kröger, 1993
). Since it is highly unlikely that the concentration of sulfide available in the plates to initiate polysulfide formation is anywhere near 10 mM, the concentration of free polysulfide in the pH 6·0 plates that could act as a potential Phs substrate will be a great deal less than 10 µM. We infer that a polysulfide reduction mechanism alone cannot account for the sulfur-clearing phenotype.
Our observations suggest that Salmonella species are capable of utilizing elemental sulfur as an electron acceptor in natural environments. They also raise the possibility that a thiosulfate cycling system could mediate sulfur respiration in other organisms. It is notable that both the thiosulfate cycling system and the polysulfide-based reactions provide a mechanism by which a substrate that cannot cross the outer membrane can be metabolized by means of reactions taking place at the cytoplasmic membrane (Fig. 4).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barrett, E. L. & Clark, M. A. (1987). Tetrathionate reduction and production of hydrogen sulfide from thiosulfate. Microbiol Rev 51, 192-205.
Barrett, E. L. & Riggs, D. L. (1982). Evidence for a second nitrate reductase activity that is distinct from the respiratory enzyme in Salmonella typhimurium. J Bacteriol 150, 563-571.[Medline]
Casadaban, M. J. & Cohen, S. N. (1979). Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci USA 76, 4530-4533.[Abstract]
Clark, M. A. & Barrett, E. L. (1987). The phs gene and hydrogen sulfide production by Salmonella typhimurium. J Bacteriol 169, 2391-2397.[Medline]
Craske, A. & Ferguson, S. J. (1986). The respiratory nitrate reductase of Paracoccus denitrificans. Molecular characterization and kinetic properties. Eur J Biochem 158, 429-436.[Abstract]
Fong, C.-L. W., Heinzinger, N. K., Tongklan, S. & Barrett, E. L. (1993). Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction. J Bacteriol 175, 6368-6371.[Abstract]
Hedderich, R., Klimmek, O., Kröger, A., Dirmeier, R., Keller, M. & Stetter, K. O. (1998). Anaerobic respiration with sulfur, polysulfide and disulfide. FEMS Microbiol Rev 22, 353-381.
Heinzinger, N. K., Fujimoto, S. Y., Clark, M. A., Moreno, M. S. & Barrett, E. L. (1995). Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism. J Bacteriol 177, 2813-2820.[Abstract]
Hensel, M., Hinsley, A. P., Nikolaus, T., Sawers, G. & Berks, B. C. (1999). The genetic basis of tetrathionate respiration in Salmonella typhimurium. Mol Microbiol 32, 275-288.[Medline]
Hille, R. (1996). The mononuclear molybdenum enzymes. Chem Rev 96, 2757-2816.[Medline]
Huang, C. J. & Barrett, E. L. (1990). Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium. J Bacteriol 172, 4100-4102.[Medline]
Kelly, D. P. & Wood, A. (1994). Synthesis and determination of thiosulphate and polythionates. Methods Enzymol 243, 475-501.
Khangulov, S. V., Gladyshev, V. N., Dismukes, G. C. & Stadtman, T. C. (1998). Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer. Biochemistry 37, 3518-3528.[Medline]
Klimmek, O., Kröger, A., Steudel, R. & Holdt, G. (1991). Growth of Wolinella succinogenes with polysulphide as terminal acceptor of phosphorylative electron transport. Arch Microbiol 155, 177-182.
Klimmek, O., Kreis, V., Klein, C., Simon, J., Wittershagen, A. & Kröger, A. (1998). The function of the periplasmic Sud protein in polysulfide respiration of Wolinella succinogenes. Eur J Biochem 253, 263-269.[Abstract]
Maloy, S. R., Stewart, V. J. & Taylor, R. K. (1996). Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Moser, D. P. & Nealson, K. H. (1996). Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur reduction. Appl Environ Microbiol 62, 2100-2105.[Abstract]
Pollock, M. R. & Knox, R. (1943). Bacterial reduction of tetrathionate. Biochem J 37, 476-481.
Pope, N. R. & Cole, J. A. (1982). Generation of a membrane-potential by one of two independent pathways for nitrite reduction by Escherichia coli. J Gen Microbiol 128, 219-222.[Medline]
Price-Carter, M., Tingey, J., Bobik, T. A. & Roth, J. R. (2001). The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar Typhimurium on ethanolamine or 1,2-propanediol. J Bacteriol 183, 2463-2475.
Riggs, D. L., Tang, J. S. & Barrett, E. L. (1987). Thiosulfate reductase as a chlorate reductase in Salmonella typhimurium. FEMS Microbiol Lett 44, 427-430.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schauder, R. & Kröger, A. (1993). Bacterial sulfur respiration. Arch Microbiol 159, 491-497.
Received 27 May 2002;
revised 15 August 2002;
accepted 16 August 2002.
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