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Keywords:

  • thiosulfate oxidation;
  • hydrogen sulfide oxidation;
  • SoxF;
  • flavocytochrome c

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Paracoccus pantotrophus strain GBsoxFΔ carries a deletion in the soxF gene that inactivates flavoprotein SoxF-sulfide dehydrogenase. This strain grew with thiosulfate slower than the wild type. GBsoxFΔ cells oxidized thiosulfate at a rate of 40% and hydrogen sulfide at a rate of 45% of the wild type. Complementation of GBsoxFΔ with plasmid pRIsoxF carrying the soxF gene increased these rates to 83% and 70%, respectively. However, GBsoxFΔ and GBsoxFΔ (pRIsoxF) oxidized thiosulfate and hydrogen sulfide to sulfate as evident from the yield of electrons. The thiosulfate oxidation rate of cell-free extracts of strain GBsoxFΔ was increased when supplemented with SoxF isolated from the wild type. However, SoxF did not affect the thiosulfate-oxidizing activity of the Sox enzyme system as reconstituted from the ‘as-isolated’ four Sox proteins. These data demonstrated that SoxF enhanced chemotrophic thiosulfate oxidation in vivo and acted on some component or condition present in whole cells and cell-free extracts but not present in the reconstituted system.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

The aerobic bacterium Paracoccus pantotrophus grows chemoautotrophically with thiosulfate and heterotrophically with various carbon sources (Robertson & Kuenen, 1983; Ludwig et al., 1993; Rainey et al., 1999; Friedrich et al., 2001).

Thiosulfate oxidation of P. pantotrophus yields eight electrons in vivo and in vitro (Friedrich et al., 2001). The sox gene cluster of P. pantotrophus encodes the sulfur oxidizing (Sox) enzyme system and comprises 15 genes which are similarly present in other chemo- and phototrophic bacteria (reviewed in Friedrich et al., 2001, 2005). Seven sox genes encode polypeptides which form four periplasmic proteins, SoxYZ, SoxXA, SoxB and SoxCD. These proteins reconstitute the Sox enzyme system and catalyze together hydrogen sulfide-, sulfur-, thiosulfate-, and sulfite-dependent horse heart cytochrome c reduction (Rother et al., 2001). The sulfur substrate is covalently attached to a sulfhydryl of a conserved cysteine residue of the SoxY protein (Quentmeier & Friedrich, 2001). This reaction is proposed to be performed by the cytochrome complex SoxXA (Friedrich et al., 2001), the crystal structure of which has been resolved recently (Dambe et al., 2005). In addition, P. pantotrophus harbors a membrane-bound sulfide : quinone oxidoreductase (Schütz et al., 1998), and in vitro hydrogen sulfide is oxidized by the flavoprotein SoxF (Quentmeier et al., 2004); both reactions yield only two electrons (Schütz et al., 1998; Quentmeier et al., 2004).

SoxF (42 797 Da) is a monomeric, FAD-containing periplasmic protein. SoxF is not required by the Sox enzyme system to oxidize the various sulfur substrates (Rother et al., 2001). SoxF of P. pantotrophus is closely related to the flavoprotein subunits of flavocytochromes c of phototrophic and chemotrophic sulfur-oxidizing bacteria (Quentmeier et al., 2004). Flavocytochromes c, the complexes of flavoproteins with a small c-type cytochrome, have been characterized from the phototrophic purple sulfur bacterium Allochromatium vinosum (formerly Chromatium vinosum; Imhoff et al., 1998) and subsequently from other phototrophic and chemotrophic bacteria (reviewed in Friedrich et al., 2005). Flavocytochromes c (FCSD) exhibit in vitro sulfide dehydrogenase activity with horse heart cytochrome c as electron acceptor and sulfur as the proposed product (Cusanovich et al., 1991). This in vitro activity has been taken as evidence for its physiological hydrogen sulfide-oxidizing role in phototrophic and chemotrophic sulfur-energy metabolism of sulfur-oxidizing bacteria (Kusai & Yamanaka, 1973; Fukumori & Yamanaka, 1979; Brune, 1989; Cusanovich et al., 1991). However, disruption of the cytochrome subunit FCSD of A. vinosum does not affect phototrophic growth with hydrogen sulfide (Reinartz et al., 1998), which questions a crucial function of FCSD in vivo. Also, in the phototrophic purple nonsulfur bacterium Rhodobacter capsulatus (DSMZ 155), a SoxF homologous flavoprotein is encoded by ORF443 which, however, does not function in sulfide oxidation in vivo. This strain lacks other sox genes and the sqr gene encodes a periplasmic sulfide : quinone oxidoreductase (SQR). Inactivation of the sqr gene disables the capacity of R. capsulatus to phototrophically oxidize and to grow with hydrogen sulfide (Schütz et al., 1999).

Here, we report that SoxF of P. pantotrophus enhances chemotrophic thiosulfate and hydrogen sulfide oxidation in vivo. In mutants unable to produce a functional SoxF protein, the yields of electrons from thiosulfate and hydrogen sulfide are similar to those of the wild type. However, homogeneous SoxF of the wild type enhances the thiosulfate oxidation rate of extracts of the mutants devoid of SoxF. These data suggest that SoxF acts on the proteins of the Sox enzyme system, rather than oxidizing thiosulfate or hydrogen sulfide in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Bacterial strains, plasmids and growth conditions

Bacterial strains used in this study are listed in Table 1. Escherichia coli was transformed as described (Chung et al., 1989). Escherichia coli S17-1 (Simon et al., 1983) was used to mobilize plasmids into Paracoccus pantotrophus GB17.

Table 1.   Bacterial strains and plasmids used in this study
Strain or plasmidRelevant genotype or phenotype*Reference or source
  • *

    Sox, chemotrophic growth with thiosulfate.

Escherichia coli
 S17-1RecA pro thi hsdS, RP4-tra-functions supE44Simon et al. (1983)
Paracoccus pantotrophus
 GB17Sox+Robertson & Kuenen (1983)
 GBΩXSox, KmrsoxX::KmΩBardischewsky et al. (2005)
 GBsoxFΔSox+, in-frame deletion in soxFRother et al. (2001)
Plasmids
 pRI1Cmr, Kmr, Mob+Pfitzner et al. (1998)
 pSOXF5N5 kb NotI fragment containing soxE′FGH in pBluescript SK(+)Rother et al. (2001)
 pRIsoxF2.8 kb PstI–XbaI fragment containing soxE′FGH′ from pSOXF5N in pRI1This study
 pVKB98911 bp HindIII fragment containing soxR‘SVWXYZABCDE’ in pVK101Rother et al. (2001)

For complementation of strain GBsoxFΔ carrying a deletion in soxF, plasmid pRIsoxF was constructed. The 2.8 kb PstI–XbaI soxE′FGH′ fragment of pSOXF5N carrying a 5 kb sox gene region (Rother et al., 2001) was cloned into plasmid pRI1 (Pfitzner et al., 1998). The plasmid was transformed in E. coli S 17-1, and conjugated therefrom into strain GBsoxFΔ to give GBsoxFΔ (pRIsoxF).

The DNA sequence of the sox gene region is accessible at the GenBank data library under accession number X79242. Paracoccus pantotrophus strains were cultivated aerobically at 30°C. The Sox enzyme system and SoxF were induced when cells were cultivated either chemoautotrophically with 20 mM thiosulfate or mixotrophically with succinate plus thiosulfate (Bardischewsky & Friedrich, 2001). For mass production of chemotrophically grown cells, P. pantotrophus was cultivated in a 300 L fermentor and cells were harvested as described (Friedrich et al., 2000).

Cell-free extracts and purification of SoxF

Cells grown mixotrophically were passed twice through a French pressure cell at 150 MPa and subjected to differential centrifugation as described (Quentmeier et al., 2000). The 200 000 g supernatant was subjected to ammonium sulfate fractionation. Proteins precipitating between 44% and 65% ammonium sulfate saturation were dialyzed against 25 mM sodium-potassium phosphate buffer, pH 6.5, and designated cell-free extracts (Friedrich et al., 2000).

SoxF was identified by immunoblot analysis and sulfide dehydrogenase activity. SoxF was purified from cell-free extracts to homogeneity by column chromatography on Q Sepharose, Resource Q, and hydroxyapatite as detailed previously (Quentmeier et al., 2004).

Enzyme assays

The thiosulfate-dependent oxygen uptake rate of whole cells was determined with a polarographic oxygen electrode in a stirred assay (3.0 mL) as described (Wodara et al., 1997).

The thiosulfate and hydrogen sulfide oxidation rate in vitro was determined by following the reduction of horse heart cytochrome c at 550 nm with a Shimadzu UV160A UV/VIS spectrophotometer (Tokyo, Japan) in an assay with an initial concentration of 10 μM of the sulfur substrate at 30°C as described previously (Rother et al., 2001).

Hydrogen sulfide-dependent activity of SoxF was determined as described previously (Quentmeier et al., 2004).

One unit (U) of enzyme activity is defined as the reduction of 1 μmol of oxygen or 1 μmol of horse heart cytochrome c per minute at 30°C.

Protein was quantified by the method of Bradford (1976).

Analytical procedures

The molecular mass of denatured proteins was determined by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) according to Laemmli (1970). Proteins were stained with Coomassie blue as described (Weber et al., 1972).

Immuno (Western) blots (Towbin et al., 1979) were performed according to the ‘semi-dry’ procedure using the multiphor electrophoresis system (Pharmacia, Freiburg, Germany). Antibodies against the oligopeptide of the highly immunogenic epitope of SoxF LDPKDKFSKQALFE (OP-F) were raised in rabbits at the facilities of Eurogentec (Seraing, Belgium). The signal intensities of the stains of the Western blots were quantified using the Scion Image software.

Genetic techniques

Standard DNA techniques were applied (Sambrook et al., 1989). Plasmid DNA was isolated according to Kieser (1984).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

SoxF enhances thiosulfate oxidation of strain GBsoxFΔ

The homogenote mutant Paracoccus pantotrophus GBsoxFΔ carries an in-frame deletion of 94 amino acids in soxF which eliminates the conserved flavin-binding site. This deletion was proposed to inactivate the enzyme (Rother et al., 2001). Strain GBsoxFΔ was re-examined for its ability to grow chemoautotrophically with thiosulfate and carbon dioxide. Strain GBsoxFΔ grew with a specific growth rate of 0.062 h−1 (doubling time td of 11.2 h) which was about half that of the wild-type 0.126 h−1 (td 5.5 h; data not shown). When P. pantotrophus strains were cultivated mixotrophically with succinate plus thiosulfate, the thiosulfate-dependent oxygen uptake rate of whole cells reached a maximum after exhaustion of succinate. The maximum activity of strain GBsoxFΔ was 57% that of the wild type, and in the stationary phase the activity decreased significantly (Fig. 1a).

image

Figure 1.  Thiosulfate oxidation rate and SoxF concentration in different Paracoccus pantotrophus strains. Cells were cultivated with 20 mM succinate plus 20 mM thiosulfate. (a) Thiosulfate-oxidizing activity was determined from 0.45 mg of protein of whole cells per assay using an oxygen electrode as described in the Materials and methods section. (▴) wild-type; (▵) strain GBsoxF; (◆) strain GBsoxFΔ (pRIsoxF). (b) Immunochemical analysis of SoxF antigens from cell-free extracts of P. pantotrophus. Cell-free extract (20 μg of protein) was applied to each well and subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis. SoxF antigens were detected by immunoblot analysis. Lane 1, P. pantotrophus wild type; lane 2, strain GBsoxFΔ; lane 3, strain GBsoxFΔ (pRIsoxF). The arrow indicates the time of sampling for Western blot analysis.

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To further examine the effect of SoxF in vivo, plasmid pRIsoxF carrying the soxE′FGH′ genes was constructed and conjugated into strain GBsoxFΔ for trans complementation of soxF, yielding strain GBsoxFΔ (pRIsoxF). This strain expressed a maximum specific thiosulfate-dependent oxygen uptake rate of 0.56 U mg−1 of protein which was about 75% of the wild-type activity and about 30% higher than strain GBsoxFΔ (Fig. 1a). Strain GBsoxFΔ (pRIsoxF) produced SoxF at a concentration of about 20% of wild-type levels, as evident from immunochemical analysis with SoxF-specific antibodies (Fig. 1b) and quantification of signal intensity by Scion image analysis. Thus, the higher thiosulfate oxidation rate was attributed to SoxF as it correlated with the higher concentration of SoxF. The comparison of the phenotypes of the wild type with strain GBsoxFΔ and GBsoxFΔ (pRIsoxF) demonstrated that SoxF was not essential for thiosulfate oxidation but enhanced the thiosulfate oxidation rate.

Paracoccus pantotrophus cells oxidize hydrogen sulfide when chemotrophically or mixotrophically precultivated with thiosulfate (Chandra & Friedrich, 1986). In strain GBsoxFΔ, the thiosulfate and hydrogen sulfide oxidation rates decreased to 40% and 45%, respectively, of the wild-type level. Both activities were increased in strain GBsoxFΔ (pRIsoxF) compared with strain GBsoxFΔ, but reached only 83% and 70% of the wild-type level (Table 2).

Table 2.   Thiosulfate and hydrogen sulfide oxidation rates and yields of electrons of Paracoccus pantotrophus cells
StrainSpecific oxygen uptake rateOxygen consumption
(μmol O2 min−1 mg of protein−1)(μmol O2 μmol of substrate−1)*
ThiosulfateHydrogen sulfideThiosulfateHydrogen sulfide
  • *

    The numbers in parentheses give the yield of electrons with the respective substrates. These yields were calculated according to the absolute amount of oxygen consumed divided by the absolute amounts of thiosulfate and sodium sulfide.

Wild-type GB170.5620.4471.96 (7.8)1.60 (6.4)
GBsoxFΔ0.2260.2031.80 (7.2)1.47 (5.9)
GBsoxFΔ(pRIsoxF)0.4690.3131.88 (7.5)1.47 (5.9)

From the wild type, the electron yield was 7.8 electrons per mol of thiosulfate and close to the theoretical yield (8.0 electrons per mol of thiosulfate). Also, in strain GBsoxFΔ, the yield was 7.2 electrons per mol of thiosulfate. Surprisingly, the electron yield from the oxidation of hydrogen sulfide by strain GBsoxFΔ was close to that determined from the wild type (Table 2). Thus, the absence of SoxF decreased both the thiosulfate and the hydrogen sulfide oxidation rates, and the presence of SoxF increased these rates while the degree of oxidation of both sulfur substrates was not affected.

Although the flavoprotein SoxF oxidizes hydrogen sulfide in a two-electron step to sulfur or polysulfide in vitro (Quentmeier et al., 2004), these results question its function as an enzyme directly involved in sulfide-dependent energy metabolism in vivo. This view is supported by the fact that hydrogen sulfide is not a free intermediate of the reaction cycle of protein-bound thiosulfate oxidation (Quentmeier & Friedrich, 2001).

SoxF enhances thiosulfate oxidation in vitro

Cell-free extracts of the wild type are able to perform thiosulfate-dependent horse heart cytochrome c reduction (Quentmeier et al., 2000) (Table 3). The addition of 0.25–2 μM of homogeneous SoxF to assays with extracts of the wild type cultivated chemolithoautotrophically with thiosulfate increased the thiosulfate-dependent cytochrome c reducing activity marginally by at most 16% as compared to without addition of SoxF (data not shown). Thus, the marginal increase did not correlate with the eightfold concentration of SoxF, which suggests that SoxF did not participate directly in thiosulfate oxidation. The thiosulfate-dependent cytochrome c reducing activity of cell-free extracts of strain GBsoxFΔ cultivated mixotrophically with succinate and thiosulfate was only 42% of the wild-type activity, and addition of 0.25 μM homogeneous SoxF to the extract of GBsoxFΔ increased the rate by 103% (Table 3). Addition of up to 2.0 μM SoxF to assays with extracts of GBsoxFΔ increased the thiosulfate oxidation rate to up to 247% (data not shown). SoxF alone was unable to perform thiosulfate-dependent reduction of horse heart cytochrome c (Table 3). Therefore, the enhancement of the thiosulfate-oxidizing activity by SoxF appeared not to be due to an involvement in thiosulfate conversion but suggested a function in the overall Sox enzyme system.

Table 3.   Thiosulfate-dependent cytochrome c reduction rates of the 44–65% ammonium sulfate-fraction of strains of Paracoccus pantotrophus devoid of SoxF and their SoxF supplementation
Cell-free extractSpecific thiosulfate-oxidizing activity (nmol cytochrome c reduced min−1 mg of protein−1)
Without additionWith 0.25 μM SoxF
  • *

    The Sox-enzyme system was reconstituted from homogeneous preparations of each 0.5 μM SoxXA, SoxYZ, SoxB and Sox(CD)2.

None00
GB171.731.35
GBsoxFΔ0.731.48
GBsoxFΔ(pRIsoxF)1.372.26
GBΩX00
GBΩX(pRIsoxF)00
GBΩX(pVKB9)1.222.23
Sox-enzyme system*18.517.2

Strain GBΩX is unable to express the sox structural genes and is thus unable to oxidize thiosulfate (Bardischewsky et al., 2005). Also, this strain is unable to oxidize hydrogen sulfide (data not shown). Therefore, it was not surprising that no thiosulfate–cytochrome c reduction was determined from extracts of strain GBΩX (Table 3). When strain GBΩX was complemented in trans with the soxF gene by transfer of plasmid pRIsoxF, SoxF was produced at a concentration comparable to that of strain GBsoxFΔ (pRIsoxF) (data not shown). However, whole cells of strain GBΩX (pRIsoxF) did not form thiosulfate- or hydrogen sulfide-oxidizing activity (data not shown) nor were its cell-free extracts able to catalyze thiosulfate-dependent cytochrome c reduction (Table 3). None of the Sox proteins required to reconstitute the thiosulfate-oxidizing enzyme system catalyze an activity that can be determined separately. SoxF exhibits sulfide dehydrogenase activity which, however, is not required for hydrogen sulfide oxidation of the reconstituted system (Rother et al., 2001).

Strain GBΩX was complemented with plasmid pVKB9 which harbors the sox structural genes excluding soxF. The thiosulfate-oxidation rate of cell-free extracts of strain GBΩX(pVKB9) was 70.5% that of the wild type and addition of SoxF to these extracts increased the rate to 183% (Table 3). Again, these data demonstrated a specific function of SoxF in thiosulfate oxidation in vitro. SoxF increased the thiosulfate-oxidizing ability in both strains to a similar degree and activity, and the identity of the data of the strains GBsoxFΔ and GBΩX(pVKB9) demonstrated that the 94 amino-acid deletion in SoxF completely eliminated its function in vivo and in vitro.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

We present here physiological and biochemical evidence that the flavoprotein SoxF functions in thiosulfate oxidation in vivo and in vitro. The physiological function of SoxF in chemotrophic thiosulfate oxidation of Paracoccus pantotrophus is evident from the decreased thiosulfate oxidation rate of strains devoid of a functional SoxF protein such as strains GBsoxFΔ and GBΩX(pVKB9). Genetic complementation of both strains with soxF increased the thiosulfate oxidation rate. The fact that this activity was lower in strain GBsoxFΔ (pRIsoxF) than in the wild type was attributed to the lower concentration of SoxF in this strain. Moreover, SoxF affects the hydrogen sulfide oxidation rate similarly to the thiosulfate oxidation rate, as is evident from the similar ratios of the in vivo activities. However, both substrates were completely oxidized to sulfate as evident from the electron yield. From these results we conclude that SoxF affected the activity of the sulfur-oxidizing enzyme system rather than the mechanism of thiosulfate and hydrogen sulfide oxidation. SoxF is not essential but beneficial for sulfur oxidation, and this implies that SoxF is not directly involved in the catalytic reaction of sulfur oxidation as performed by the Sox enzyme system in vivo and in vitro. SoxF may, therefore, act either on the protein(s) involved in sulfur oxidation or on a condition present in the cells and cell-free extracts but not in the Sox enzyme system reconstituted from homogeneous proteins.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

We thank B. Höller and J. Ringk for excellent technical assistance. Financial support of this study by the Deutsche Forschungsgemeinschaft (Fr 318/9-1) to C.G.F. is gratefully acknowledged.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
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