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Journal of Bacteriology, February 2007, p. 851-859, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01080-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Thiol:Disulfide Oxidoreductase DsbB Mediates the Oxidizing Effects of the Toxic Metalloid Tellurite (TeO32–) on the Plasma Membrane Redox System of the Facultative Phototroph Rhodobacter capsulatus{triangledown}

Francesca Borsetti,1 Francesco Francia,1 Raymond J. Turner,2 and Davide Zannoni1*

Department of Biology, University of Bologna, Bologna, Italy,1 Department of Biological Sciences, University of Calgary, Calgary, Canada2

Received 21 July 2006/ Accepted 31 October 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The highly toxic oxyanion tellurite (TeO32–) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbBWT, in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an "electron conduit" between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of TeIV, the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the environment, tellurium (Te) exists in its elemental—Te0—inorganic—telluride (Te2–), tellurite (TeO32–), and tellurate (TeO42–)—and organic—dimethyl telluride (CH3TeCH3)—forms (8). Of these, the toxic oxyanion forms, TeO32– and TeO42–, are more common than and are highly soluble compared to nontoxic elemental tellurium, Te0 (38). Tellurium is widely used in the electronics industry, for photoreceptors, thermocouples, and batteries, but also in metallurgical processes and as an additive to industrial glasses (8). As a result, microorganisms are now becoming exposed to abnormal concentrations of this element, and bacterial species resistant to tellurium can easily be isolated from industrial sludge (38). However, research into the anthropogenic emission of Te-based compounds is scarce, and the implications for selection of microorganisms resistant to tellurite (TeO32–) and tellurate (TeO42–) are largely unexplored (40).

Tellurite is more toxic to mammalian cells (43) and microorganisms (38) than are several heavy metals, e.g., mercury, cadmium, zinc, chromium, and cobalt, which are objects of major public health concern (38). Depending on the strain, the concentration of tellurite inhibiting microbial growth ranges from 1 to 1,000 µg/ml (34, 38, 46-48). Microorganisms counteract tellurite (TeO32–) toxicity in several ways, namely, by (i) decreasing its uptake, (ii) enhancing its efflux, or (iii) chemically modifying it through methylation or reduction to the less toxic elemental tellurium (Te0) (8). The latter strategy of detoxification is present in the bacterial genera that are phenotypically characterized by cell darkening due to intracellular accumulation of black inclusions of Te0 (4, 30, 38, 46-48), although tellurite resistance (Ter) does not strictly depend on the formation of Te0 (46-48).

The mechanism of tellurite reduction by microorganisms remains unclear, although it has been extensively discussed in the literature (8, 38-41, 46-48). In Escherichia coli, the thiol:redox buffering enzymes (glutathione and thioredoxin reductases) and their metabolites (thioredoxin, glutaredoxin, and glutathione) were suggested to be involved in tellurite reduction (40), while the membrane-bound nitrate reductases NarG and NarZ have been found to reduce tellurite (1). Cells of the facultative phototroph Rhodobacter sphaeroides accumulate Te0 crystallites inside the internal membrane system (30-31); accordingly, it was suggested that the plasma membrane redox chain might have a role in tellurite reduction, as it was also dependent on reduced flavin dinucleotide oxidation activity (30-31). The reduction of tellurite by chemotrophically grown cells of E. coli, Erwinia carotovora, and Agrobacterium tumefaciens has been related to the activity and membrane location and sidedness of the respiratory cytochrome oxidases (Cox), although the stimulation of Cox activity in cells of P. aeruginosa was seen to lower the cell Te0 content (39). The latter evidence is clearly in contrast with a role of Cox in TeO32– reduction but conversely is in line with other reports indicating that Cox activity in cells of Pseudomonas pseudoalcaligenes KF707 and Rhodobacter capsulatus grown in the presence of tellurite drops in parallel with a cytosolic accumulation of Te0 and a drastic decrease of the c-type cytochrome (Cyt c) content (4, 17). These observations led Borsetti et al. (4) to suggest that the respiratory Cox oxidases of R. capsulatus and P. pseudoalcaligenes were not involved in the reduction of tellurite to Te0. On the other hand, the question of whether the Cyt c-type pool modifications in cells of P. pseudoalcaligenes KF707 and R. capsulatus are due to TeO32– toxicity on c-type heme assembly and/or expression or whether they reflect a direct toxic effect of the oxyanion on the periplasmic facing proteins remains unsolved (however, see below). Here, we have addressed this question by studying the interaction of tellurite with isolated membranes from R. capsulatus. We report that under steady-state respiratory conditions, the oxidation-reduction levels of both Cyt b and Cyt c are increased by tellurite. In line with this, the rereduction of Cyt c which follows its photooxidation by a series of actinic flashes of light is accelerated by tellurite. This phenomenon is blocked by the bc1 complex specific inhibitor antimycin A, and it is absent from membranes of R. capsulatus MD22, a mutant lacking the membrane-bound thiol:disulfide oxidoreductase DsbB. These data were interpreted to show that tellurite, a pro-oxidant agent in intact cells, alters the redox equilibrium of the Q/QH2-bc1-c2/cy segment of the redox chain. The tellurite effect would be mediated by the thiol:disulfide oxidoreductase DsbB, a possible redox partner of the Q pool, as suggested for E. coli (21). Our finding is therefore in contrast with the most accepted concept that tellurite would act as a general oxidant (38). Conversely, our data give strong experimental support and molecular evidence to early indications by Moore and Kaplan (31) that under specific growth conditions and tellurite concentrations, the oxyanion might act as a disposal sink for the excess of reducing power at the Q-pool level of photosynthetic facultative phototrophs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and cell growth. The strains used are listed in Table 1, along with their relevant properties. R. capsulatus MT1131 (wild type [WT]) and the mutant strains MD22 (DsbB–), MD22/pDsbBWT (DsbB+), MT1131/pDsbBWT (DsbB overexpressed), FJ1 (Cyt cy–), and MT-G4/S4 (Cyt c2–) were grown under phototrophic conditions in screw-cap tubes and bottles (30°C) with an incident light intensity of 200 W m–2. The M6G (Qox–) strain was grown under chemotrophic conditions (30°C) in Erlenmeyer flasks shaken at 150 rpm. The cells were cultivated in YPS (3 g/liter peptone, 3 g/liter yeast extract, 2 mM MgSO4, 2 mM CaCl2) or minimal (MedA) medium (37) supplemented with the appropriate antibiotics (kanamycin or spectinomycin, 10 µg/liter). Cell cultures were harvested in their exponential phases (optical density at 600 nm, 0.5) and kept at 4°C until membrane isolation.


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  		TABLE 1. Relevant genotypic and phenotypic features of the strains used

 
Plasma membrane isolation and protein and BChl estimations. Membrane fragments were prepared in MOPS (morpholinepropanesulfonic acid) buffer (pH 7.0) containing 5 mM KCl with a French pressure cell and differential ultracentrifugation, as reported by Zannoni et al. (53). The protein content of the samples was determined by the method of Lowry et al. (27) with bovine serum albumin as a standard. The bacteriochlorophyll (BChl) content was measured at 775 nm in acetone-methanol (7:2, vol/vol) extracts with an extinction coefficient of 75 nM–1 cm–1 (9).

Cytochrome spectral analysis. Reduced (with either NADH or sodium dithionite) minus oxidized (with potassium ferricyanide) optical difference spectra of cytochromes in membrane fragments and soluble fractions were performed at room temperature by means of a Jasco 7800 UV/VS spectrophotometer. The following extinction coefficients were used: c-type cytochromes, {varepsilon}551-540 of 19 mM–1 cm–1; b-type cytochromes, {varepsilon}561-575 of 22 mM–1 cm–1. Other assay conditions were as follows: bandwidth, 5 nm; recording time, 8.75 s; scan speed, 240 nm min–1.

Flash spectroscopy. Absorbance changes induced by a xenon flash (EG&G FX201), discharging a 3 x10–6-F capacitor charged to a 1.5-kV, 4 x 10–6-s pulse duration at half-maximal intensity, were measured by a single-beam spectrophotometer equipped with a double grating monochromator (bandwidth, 1.5 nm). The photomultiplier was protected by a Corning glass 4/96 filter, and a triggered shutter was used to gate the measuring beam. Data were acquired by a LeCroy 9410 digital oscilloscope interfaced to an Olivetti M240 computer. To determine the kinetics and the stoichiometries of the photosynthetic redox components, trains of eight flashes of light, spaced 100 ms apart, were used to photoactivate the membrane fragments in the presence of 5 µM valinomycin and 5 µM nigericin, as uncouplers and, when required, 10 µM antimycin A, as an inhibitor of the photocyclic electron flow. See below and figure legends for further details.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tellurite effects on the reduction rate of c-type cytochromes following a train of flashes of light. In recent years we have shown that membranes isolated from Rhodobacter capsulatus cells grown in the presence of the toxic oxyanion tellurite are able to catalyze a light-induced cyclic electron flow linked to the formation of an electrochemical membrane potential (4).

The carotenoid absorbance band shift is a response to transmembrane electrochemical potential, and it can be used as an internal membrane probe to show the integrity of isolated membrane vesicles in terms of proton coupling, since in the presence of uncouplers, e.g., valinomycin and nigericin, the carotenoid band shift drops dramatically due to the immediate (a few milliseconds) collapse of the membrane potential (10). In membranes of R. capsulatus MT1131 treated with 1.0 mM tellurite, the carotenoid band shift lasts for (at least) 0.1 s after an actinic flash of light (not shown); this demonstrates that the oxyanion does not act as an uncoupling agent.

The top panel of Fig. 1A shows the responses of Cyt c (examined at 551 to 542 nm) subjected to trains of eight actinic flashes of light separated by 100 ms in the absence (trace a) or presence (trace b) of 1.0 mM tellurite. Clearly, consistent Cyt c oxidation (downward signals) and rereduction (upward signals) can be seen; however, most interestingly, the rereduction kinetics are clearly faster in membranes treated with tellurite (trace b) than in control membranes (trace a).


Figure 1
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  		FIG. 1. Responses of c-type cytochromes (detected at 551 to 542 nm) in membrane fragments from light-grown cells of R. capsulatus MT1131 subjected to a series of actinic flashes of light (top and bottom traces in panel A) or to a single flash of light (traces in panel B). (A) (Top) Cyt c kinetics under multiple-turnover flashes, fired 100 ms apart, in uncoupled membranes, i.e., in the presence of valynomycin plus nigericin (5 µM each). Trace a, control; trace b, addition of 1.0 mM tellurite. (Bottom) Cyt c kinetics upon addition of 10 µM antimycin A. Traces a (control) and b (addition of tellurite) overlay, since no difference was seen between control- and tellurite-incubated samples. (B) Enlargement of the Cyt c photooxidation kinetics seen after the first flash of light. The t1/2 decays in control (t1/2a) and tellurite-treated (t1/2b) membranes are indicated. Assay conditions were as follows. Membranes, equivalent to 50 µM BChl, were suspended in 50 mM Gly-Gly buffer (pH 7.5)-20 mM KCl. Aliquots from the same membrane suspension were kept for several hours at 4°C in the absence (traces a) or presence (traces b) of TeO32–. K-cyanide (0.5 mM) and K-ascorbate (1.0 mM) were added prior to measurements to maintain the c-type hemes in a reduced state before light activation.

 
Antimycin A, which inhibits Cyt c rereduction by blocking the bc1 complex (7, 22; see also Fig. 7), is expected to reveal maximal photooxidation of these cytochromes. Indeed, as shown in the bottom panel of Fig. 1A (overlapping traces a + b), the extent of Cyt c oxidation is almost twice that seen in the absence of inhibitor (Fig. 1A, top), and it is similar in control (trace a) and tellurite-treated (trace b) membranes with no signal decay (Cyt c rereduction) after the second flash of light (200 ms). Notably, all the kinetic traces shown in Fig. 1A (top and bottom) were obtained in the presence of uncouplers in order to get the maximal rate of electron transport due to the lack of membrane potential (10).


Figure 7
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  		FIG. 7. Working models showing the possible interactions between the photosynthetic/respiratory redox carriers and the thiol:disulfide oxidoreductases DsbA/DsbB in cells of Rhodobacter capsulatus (A) and in isolated membranes treated with tellurite (B). (A) Based on the present knowledge of E. coli (32) and R. capsulatus (16), DsbA oxidizes cysteines of periplasmic proteins and delivers the reducing equivalents to the respiratory system by means of DsbB, directly interacting with the Q pool. (B) The scheme shows that in isolated membranes, the thiol:disulfide oxidoreductase DsbB mediates the oxidizing effect of TeO32– oxyanions on the redox level of the QH2:Cyt c oxidoreductase of R. capsulatus. Semiquinone (Q·–) or, alternatively, quinol (QH2) molecules are oxidized at the DsbB level (Qd site), and reducing equivalents are moved to the periplasmic space and then to TeO32– by means of the disulfide groups of DsbB (32). Generation of Q molecules would favor the oxidation of QH2 accumulated at the Qo site of the bc1 complex with a parallel reduction of the upper part of the redox chain (FeS->c1->cy/c2). See the text for further details. Symbols and abbreviations: AA, antimycin A; bL and bH, b-type hemes of the bc1 complex (7, 10); e–, electrons; FeS, Rieske iron-sulfur center; C1, Cy, and C2, cytochromes c1, cy and c2, respectively; Qo, Qi, Qd, and Qox, Q-binding sites; RC, photosynthetic reaction center; Qa, primary quinone acceptor; h{nu}, radiant energy. The question marks from DsbB to TeO32– symbolize the present lack of evidence that DsbB directly reduces the exogenously added oxyanion in intact cells.

 
To clearly show the differences in slopes of the Cyt c rereduction kinetics of MT1131 membranes, an enlargement of the Cyt c oxidation decays after the first flash of light is shown in Fig. 1B. Apparently, the half-life of Cyt c rereduction in control membranes (t1/2 {cong} 56 ± 1 ms, trace a) is twice that measured in the presence of tellurite (t1/2 {cong} 28 ± 1 ms, trace b).

To further clarify the effects induced by tellurite on the oxidation-reduction kinetics of c-type hemes, membranes treated with various concentrations of tellurite (0.25 mM, 2.5 mM) under various incubation periods (5 min, 2.5 h) were examined. The kinetic traces (not shown) indicated that the tellurite-induced acceleration of the rereduction rate was already evident in membranes incubated for 5 min with 0.25 mM tellurite.

Overall, the results presented in Fig. 1 indicate that the acceleration of the Cyt c photooxidation decay observed in tellurite-treated membranes is not due to artificial, i.e., nonphysiological, electron transfer (as shown by antimycin A inhibition) or to membrane uncoupling (as shown by the carotenoid band shift) and that c-type cytochromes are not directly affected by tellurite toxicity (see figure legends for further details).

Tellurite effects on the oxidoreduction state of b- and c-type cytochromes under steady-state respiration. Figure 2 shows the time course of the b- and c-type cytochrome oxidoreduction state as induced by NADH oxidation. The NADH-dependent electron flow raised the reduction level of c-type cytochromes to 10% ± 2% (after 2 to 3 min of respiration), while only 5 ± 1% of b-type hemes was seen under respiratory conditions (Fig. 2). Notably, the addition of tellurite (at 10 min) significantly increased the amount of both c- and b-type hemes (28% ± 3% and 20% ± 2%, respectively) reduced by NADH. Tellurite not only affected the steady-state reduction level but also increased the total amount of b- and c-type hemes reduced after the addition of 50 µM cyanide (at 16 min), a concentration inhibiting the Cyt c oxidase (Cox) activity of R. capsulatus (50-52). To explore the possibility that reduction of both b- and c-type cytochromes results from a reequilibrium of reducing equivalents along the Q-bc1-Cyt c segment of the redox chain and to exclude the possibility that it is not a specific phenomenon of photosynthetically grown cells, we tested the effect of tellurite on membranes from aerobically dark-grown cells (Fig. 3). As previously shown, the respiratory chain of R. capsulatus is branched at the Q/bc1 level, channeling reducing equivalents to a Cyt c oxidase (Cox) inhibited by 50 µM cyanide and to a quinol oxidase of the bb3 type (Qox) inhibited by 2 to 3 mM concentrations of cyanide (50). To restrict the rearrangement of reducing equivalents inside the Q/Cyt bc1/Cyt c segment of the redox chain, membranes from R. capsulatus MG6, a mutant lacking the quinol cyanide-resistant oxidase (Table 1) (19, 29), were used. Traces in Fig. 3 show a series of spectra, taken at different times, following NADH (100 µM) addition to membranes previously oxidized by the addition of a few crystals of potassium ferricyanide (dotted line). Due to the high rate of NADH consumption by aerobic membranes, a significant steady-state reduction level of c-type cytochromes is reached within a few seconds (8.7-s recording time) after substrate addition (trace a, 38% ± 3% reduction at 550 nm, 11% ± 2% at 560 nm). Upon consumption of NADH, the oxidation-reduction level at 550 nm drops from 38% ± 3% to 12% ± 1% (trace b), and this reduction level does not change in the presence of 50 µM cyanide (trace c), which confirms the complete exhaustion of NADH. At this stage, in the absence of NADH, addition of tellurite (2.5 mM) (trace d) induces a significant (30% ± 2%) and rapid Cyt c reduction which reflects the shift of reducing equivalents, accumulated as a ubiquinol (UQH2) pool, toward the upper part of the redox chain. Notably, the spectrum recorded 2 min after the addition of tellurite (trace e) shows a reduction of 80% ± 3% of the signal at 550 nm (Cyt c) and 28% ± 2% of the signal at 560 nm (Cyt b) compared to the control trace obtained in the presence of sodium dithionite (100% reduction, trace f, dashed line).


Figure 2
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  		FIG. 2. Oxidation-reduction levels of cytochromes of the c (circles) and b (squares) types in membranes from R. capsulatus MT1131 under NADH-dependent steady-state respiration as a function of time in the absence (open symbols) or presence (closed symbols) of tellurite. Additions of tellurite (2.5 mM) and cyanide (0.05 mM) are indicated by vertical arrows. Values were calculated by assuming the 100% reduction signals at 551 to 540 nm (Cyt c) and 561 to 575 nm (Cyt b) to be those obtained by adding a few crystals of sodium dithionite in a parallel assay and in the absence of tellurite. The data points represent averages of three different membrane preparations.

 

Figure 3
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  		FIG. 3. Reduced-minus-oxidized optical spectra of membranes from aerobically dark-grown cells of R. capsulatus M6G. Membranes (2.7 mg of protein/ml) were suspended in Gly-Gly buffer (pH 8.0)-5 mM KCl, and spectra were recorded at room temperature. Spectra from a to e were recorded consecutively at the following recording times: a, 0 min; b, 2 min; c, 3 min; d, 4 min; e, 6 min. Experimental conditions were as follows: trace a, 100 µM NADH; trace b, [NADH] = 0; trace c, 0.05 mM KCN; trace d, 2.5 mM tellurite; trace e, recorded 2 min after the addition of tellurite; trace f, control trace recorded after the addition of a few crystals of sodium dithionite in the absence of tellurite. The bottom dotted trace represents the baseline (fully oxidized membranes), which is obtained by the addition of a few crystals of potassium ferricyanide. See the text for further details.

 
The QH2 pool delivers reducing equivalents to the Cyt c pool (cy plus c2). The mechanism of tellurite-induced Cyt c reduction was further dissected by using membranes from FJ1 and MT-G4/S4, two mutant strains deficient in either Cyt cy or Cyt c2, respectively. As shown in Fig. 4, the c-type heme extrareduction is seen in both membranes from MT-G4/S4 (panel A) and FJ1 (panel B) mutants upon the addition of 2.5 mM tellurite. Although this phenomenon was less pronounced in membranes of MT-G4/S4 (Cyt c2–, 12 to 13% extrareduction) than in those from FJ1 (Cyt cy–, 18% extrareduction), it can be concluded that both cy and c2 hemes contribute to the Cyt c extrareduction seen in R. capsulatus MT1131 (WT) membranes (28% extrareduction) (Fig. 2). Further, the increase in the c-type reduction induced by tellurite in MTG4/S4 membranes is clearly inhibited by antimycin A (Fig. 4A) or myxothiazol (not shown). This demonstrates the physiological interaction of the toxic oxyanion with the redox chain.


Figure 4
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  		FIG. 4. Oxidation-reduction levels of Cyt c (circles) and Cyt b (squares) in membranes from R. capsulatus MTG4/S4 (Cyt c2 mutant, panel A) and R. capsulatus FJ1 (Cyt cy mutant, panel B) under NADH-dependent respiration as a function of time in the absence (open symbols) or presence (closed symbols) of tellurite. Additions of tellurite (2.5 mM) and cyanide (0.05 mM) are indicated by vertical arrows. The dotted trace between open triangles (panel A, MTG4/S4 membranes) indicates the experimental points at 551 to 540 nm (Cyt c) obtained in the presence of antimycin A (10 µM). The data points represent averages of two different membrane preparations.

 
Tellurite interaction with the Q pool involves the thiol:disulfide oxidoreductase DsbB. The precise mode of tellurite interaction with cells is not known (40). In aqueous solution, the standard reduction potential at basic pHs of the couple Te/TeO32– is 0.42 V, while at pH 7.0 it is 0.123 V. Thus, the most likely interaction, if any, would be at the Q-pool level, since the Em,7 of the redox couples Q·–/Q and Q/QH2 span from –150 to +90 mV (10). In E. coli, the membrane-bound thiol:disulfide oxidoreductase DsbB, which delivers oxidizing equivalents through DsbA to periplasmic and secreted proteins, becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains (18). To test whether or not tellurite might affect the redox equilibrium of the respiratory chain components by using DsbB as an "electron conduit" between the periplasm and the lipid-embedded Q pool, the same set of experiments as reported in Fig. 2 was performed with membranes from R. capsulatus MD22, a DsbB– mutant (16). Figure 5 clearly indicates that the addition of tellurite to MD22 membranes actively respiring NADH does not affect the redox level of either b- or c-type cytochromes. This result was further tested with cells of MD22 in which the DsbB gene was expressed from plasmid pDsbB to regain the WT phenotype. Indeed, a series of spectra taken at different times (see the experimental plan of Fig. 3) indicated that the reduction levels of the b- and c-type cytochromes were drastically increased by tellurite addition (data not shown). However, since the lack of a tellurite effect in membranes from MD22 cells might also result from a mutated respiratory phenotype, the respiratory activities of membranes from MD22 and MT1131 were compared. As detailed in Table 2, all respiratory activities tested in the presence or absence of specific inhibitors (antimycin A, myxothiazol, and cyanide) are similar in both types of membranes, reflecting also a similar cytochrome content (±22% ± 3%). These results not only confirm the original report by Deshmukh et al. (16) indicating that in R. capsulatus the dithiol:disulfide reductase DsbB is not essential for cytochrome c biogenesis but also show that DsbB is not an essential component of respiration catalyzed by membranes of R. capsulatus. Table 2 also shows that membranes from MD22/pDsbBWT cells, but also MT1131/pDsbBWT used as a control, express a cytochrome content which is considerably higher (1.9 to 2.1 times) than that present in MD22 and MT1131 membranes. As expected, the overexpression of both b- and c-type cytochromes supports a series of respiratory activities which are several times faster than those measures in MD22 and MT1131 membranes, although they are qualitatively similar in both strains. Apparently, plasmid expression of the DsbB gene in MD22 cells does not emphasize any evident deficiency of the MD22 phenotype compared to that expressed by MT1131.


Figure 5
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  		FIG. 5. Oxidation-reduction levels of Cyt c (circles) and Cyt b (squares) in membranes from R. capsulatus MD22 (DsbB mutant) under NADH-dependent respiration as a function of time in the absence (open symbols) or presence (closed symbols) of tellurite. Addition of tellurite (2.5 mM) is indicated by a vertical arrow. See the text for further details. The data points represent averages of three different membrane preparations.

 

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  		TABLE 2. Respiratory activities and cytochrome contents in membranes from R. capsulatus MT1131 (WT), MD22 (DsbB–), MD22/pDsbBWT (pDsbB+), and MT1131/pDsbBWT (pDsbB+) cellsa

 
The kinetics of Cyt c rereduction following a train of eight flashes of light in membranes from MD22, in the absence or presence of tellurite, are shown in Fig. 6A (top panel). As demonstrated by others (7), Cyt c oxidation-reduction is best explained by second-order kinetics, which indicates a concentration dependence by the substrates, i.e., soluble Cyt c and photochemical reaction center (RC). Accordingly, since MD22 membranes contain less Cyt c and photosynthetic pigments than do MT1131 membranes (see also Table 2 and the legend to Fig. 6), the c-type rereduction t1/2 is expected to be shorter than that seen in MT1131, with a higher noise/signal ratio. Indeed, as shown in Fig. 6B, which represents an enlargement of the Cyt c photooxidation decay at the first flash of light, the t1/2 of MD22 membranes is {cong}31 ± 2 ms, while the corresponding t1/2 of MT1131 is {cong}56 ± 1 ms (see Fig. 1B). However, as also shown in Fig. 6, the most significant difference between the rereduction kinetics in membranes from MT1131 and MD22 cells is that in the latter case, the apparent t1/2 of the photooxidation decay does not vary after incubation with 1.0 mM tellurite (Fig. 6B, trace b). Thus, in line with results of Fig. 5, DsbB specifically senses the tellurite effect on the electron transfer system of R. capsulatus. This conclusion is strengthened by the results of the bottom panel of Fig. 6A (trace b), showing that the Cyt c rereduction in membranes from MD22 treated with tellurite is fully sensitive to antimycin A (and myxothiazol, [not shown]), as previously shown with MT1131 WT membranes (Fig. 1A, bottom).


Figure 6
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  		FIG. 6. Responses of c-type cytochromes (551 to 542 nm) to either a series of flashes of light (A) or a single flash of light (B) in membranes from R. capsulatus MD22 (DsbB mutant) in the absence (traces a in panels A and B) or presence (traces b in panel A and B) of 1.0 mM tellurite. Trace b, bottom of panel A, shows Cyt c photooxidation kinetics after the addition of 10 µM antimycin to membranes treated with tellurite (1.0 mM). In all experiments, membranes (150 µM BChl-0.900 µM RC-0.6 µM c-type hemes) were suspended in Gly-Gly buffer (pH 7.5), 20 mM KCl, 1.0 mM K-ascorbate, 0.5 mM KCN, and valinomycin plus nigericin (5 µM each).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The facultative phototrophic bacterium Rhodobacter capsulatus is tolerant of tellurite (3) and has been shown to accumulate intracellular crystals of elemental tellurium (Te0) (4). The mechanism of cytoplasmic Te0 accumulation involves initially a {Delta}pH-dependent uptake of tellurite (5); the subsequent reduction/precipitation of Te0 made by undefined thiols as reducing agents (40) generates toxic reactive oxygen species, as we have shown that TeIV increases the activity of the superoxide dismutase (6). Thus, while the cytosolic pro-oxidant action of this toxic metalloid (40) is beginning to be understood, the mechanism behind the effect of tellurite in the periplasmic space is mostly unexplored (40).

Here, we show that tellurite added to isolated membrane fragments accelerates the rate of electron transfer through the quinol:Cyt c oxidoreductase following a series of flashes of light; furthermore, a significant c- and b-type extrareduction is seen under steady-state respiratory conditions after the addition of tellurite. These findings are clearly in contrast with previous reports on cells of P. aeruginosa, E. coli, E. carotovora, and A. tumefaciens (39), proposing a role of the terminal respiratory oxidases in the reduction of tellurite. Furthermore, TeIV-induced redox unbalance of the plasma membrane is unlikely to be caused by a direct interaction of the oxyanion with the membrane redox components because (i) the in vitro spectroscopic and biochemical features of both cytochrome c2 and ubiquinone 2 (UQ-2) in their oxidized or reduced states are not altered by tellurite (this work; also data not shown) and (ii) the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the ubiquinol:Cyt c oxidoreductase, block the effects of tellurite. It is therefore evident that the interaction of tellurite with membranes is specifically mediated by membrane-associated redox proteins and that this interaction causes a shift of reducing equivalents from the QH2-pool level to the bc1/c2-cy segment of the redox chain. Indeed, tellurite does not alter the redox equilibrium of the redox chain under fully oxidized conditions, and its effect is evident only in a situation of redox disequilibrium, i.e., when the Q pool has partially been reduced by NADH or light-dependent electron flow (as demonstrated here). The Q-cycle mechanism (7, 10, 12, 33) (schematically drawn in Fig. 7A) provides a suitable model for explaining how QH2 oxidation at the Qo site of the bc1 complex generates a concerted two-step reaction driving reduction both of bL-type heme and of the Rieske-type iron-sulfur center (FeS). One thermodynamic problem of the Q cycle is that the reduction of Q to Q·–, and then to QH2, is not favored (10), so that accumulation of QH2 would restrict the overall Q-cycle turnover. This major flow explains why the maximal activity of the photocyclic electron flow of several facultative phototrophs depends on the presence of a quinol oxidase (Qox)-containing pathway, which keeps the QH2 pool largely oxidized (49). As shown by others (23), the reduction of Q to Q·– is paralleled by reduction of Cyt c1 (Fig. 7A); thus, the oxidation of Q·– or QH2 induced by TeO32– is expected to generate a Q/QH2 ratio that is optimal for maximal Cyt c reduction under respiratory or photosynthetic conditions (see the working model in Fig. 7B, based on the results presented here). This conclusion is also fully justified by antimycin A inhibition of the process (Fig. 1, 4, and 6) and by considering that, under conditions of steady-state respiration or photosynthesis, membranes of R. capsulatus contain an excess of QH2 molecules (19, 42, 49, 50, 52).

Interestingly, we show here that the tellurite-induced shift of reducing equivalents at the Q/bc1/c2-cy level is mediated by the thiol:disulfide oxidoreductase DsbB, as this phenomenon is not seen in membranes from R. capsulatus MD22, a mutant lacking the thiol:disulfide oxidoreductase DsbB (16). The mechanism by which tellurite affects the redox equilibrium of the electron transport components through the involvement of DsbB is presently unclear (see below, however, and the working model in Fig. 7B). The reported standard reduction potential at basic pHs of the couple Te/TeO32– is –0.42 V; based on the dissociation constants of tellurous acid (3 x 10–3 and 2 x 10–8 for k1 and k2, respectively), tellurite at pH 7.0 should be present in the form HTeO3– and TeO32– (104:1 ratio), with no TeIV present due to its instability in water. We know from work done with E. coli (21) that DsbB is predicted to contain four transmembrane stretches (32) and two periplasmic loops, each of which contains a pair of cysteines. Reduced DsbB shows an in vitro Q-reductase activity (2, 35), as a molecule of hydroquinone is suggested to be tightly bound to DsbB whereas a second Q molecule would be exchangeable with the rest of the Q pool (Em,7 = +90 mV) (11). Unfortunately, no redox potential is available for the Q at the DsbB interaction site of E. coli (20, 36), while the DsbB cysteines were titrated at –70 mV (Cys41-Cys44) and –186 mV (Cys104-Cys130) (18). Assuming that E. coli and R. capsulatus contain DsbB with similar redox properties and structural features (16), the standard potential (Eo') of the HTeO3–/TeO32– couple free to react with DsbB cysteines would be close to –0.12 V (44), which would appear to make it not suitable for the oxidation of Cys41-Cys44 disulfide (Em,7 = –0.07 V). However, the Cys104-Cys130 disulfide (Em,7 = –0.186 V) is much less oxidizing than the toxic metalloid, and this would drive the overall DsbB oxidation to completion, being also favored by an estimated TeIV/DsbB ratio of 106 under the experimental conditions reported here (see Materials and Methods). One prerequisite for this mechanism to occur is that the Michaelis constant, Km, for Q/ Q·– at the Qo site of the bc1 complex is less than or equal to the Km for Q/ Q·– at the Qd-binding site of the DsbB protein (see the working model in Fig. 7B). The DsbB isolated from E. coli has an in vitro Km of {cong}2 µM for benzoquinone (QoC10) (2), while the Km of UQH2 for bc1 complexes isolated from different sources varies from 8 to 20 µM and from 1 to 4 with ubiquinol-1 and ubiquinol-2 as substrates, respectively (24-25). Apparently, these in vitro values of Km for isolated bc1 complexes are compatible with the Km reported for E. coli DsbB, even though it would be premature to reach a firm conclusion on this point by simply comparing the Km of quinones and quinols having different hydrocarbon chain lengths. Additionally, although the above-reported considerations provide a thermodynamic explanation for the tellurite interaction at the DsbB/Q/bc1 level of the redox chain in isolated membranes of R. capsulatus, more experiments are required to establish whether the membrane-associated thiol:disulfide oxidoreductase DsbB is the actual direct electron donor to periplasmically located tellurite in intact cells or other unknown components are involved.

Preliminary results indicate that membranes from MD22/pDsbBWT and MT1131/pDsbBWT, in which the DsbB protein is overexpressed (Table 2), catalyze a significant tellurite reduction, as indicated by darkening at 540 nm (0.7 {Delta}A min–1 mg of protein–1). This finding suggests that DsbB might function as the catalytic part of a QH2:TeO32– oxidoreductase event under suitable redox conditions. Indeed, although no dissimilatory electron transport to tellurite is known to date (47), the energy of the TeO32–/Te redox couple would be in principle more favorable for anaerobic respiration than would the SO42–/HS– redox couple (–0.217 V) (26) that is widely utilized by sulfate reducers. Perhaps the cytosolic toxicity of tellurite would restrict the dissimilatory utilization of this oxyanion by microorganisms, as the anaerobic respiration of the less-toxic tellurate (TeO42–) in deep ocean bacteria has recently been reported (13). Additionally, we have shown that R. capsulatus MT1131 cell viability, as detected on agar plates, is unaffected by 1.6 to 4.0 mM tellurite (6). This indicates that the experimental conditions used in the present work are not far from those faced by intact cells in the presence of large amounts of tellurite, e.g., industrially contaminated areas (45).

At the present experimental stage, our data would exclude a role of both quinol and cytochrome oxidases in TeIV reduction by membranes of R. capsulatus, as suggested for E. coli, E. carotovora, and A. tumefaciens (39). In line with our conclusion is the observation that the TeIV reduction capability of Shewanella oneidensis was not affected by the lack of the membrane-bound tetracytochrome c CymA (23). Further, TeIV reduction was inhibited only by 2-n-hepthyl-4-hydroxyquinoline-N-oxide and antimycin A, suggesting the involvement of the Q/bc1 segment of the redox chain (23). The latter result and those reported in the present work give strong experimental support to an early indication by Moore and Kaplan (31) that photosynthetic bacteria might use various metalloids as potential electron sinks under particular reducing growth conditions, such as, for example, anaerobic photoheterotrophy in aquatic polluted sites.

An aspect which is presently poorly understood concerns the role of the thiol:disulfide oxidoreductases DsbA and/or DsbB, in both the Cyt c biogenesis and tolerance to tellurite of R. capsulatus, as the amount of c-type hemes drastically drops in membranes from cells grown in the presence of tellurite (4) and DsbB can be a redox partner of tellurite (this work). It has been shown that R. capsulatus mutants lacking either DsbA or DsbB are fully proficient in anaerobic photosynthetic growth and are able to produce similar amounts of c-type cytochromes (16). Conversely, the DsbB-deficient mutant MD22 is proficient in respiratory metabolism in both enriched and minimal growth media, while the DsbA mutant MD20 is impaired in respiration only in enriched growth medium (16). This would suggest that the lack of the periplasmic thiol:disulfide oxidoreductase DsbA has a stronger effect on the respiratory metabolism of R. capsulatus than does the lack of the plasma membrane-bound DsbB (see also Table 2), a possibility which is in conflict with the present models. Preliminary results indicate that the tellurite MIC for MD20 cells is four to five times lower than the MICs for WT and MD22 mutant cells (unpublished results). These findings suggest that DsbA, but not DsbB, is required for growth in the presence of tellurite because, based on the results presented here, DsbB would be the key site of tellurite interaction. Indeed, the low tolerance to tellurite of MD20 might indicate that the lack of DsbA may leave DsbB "open" to periplasmic redox injuries, e.g., large amounts of metalloids.


   ACKNOWLEDGMENTS
 
This work was supported by MIUR (PRIN 2005); R.J.T. is supported by NSERC, Canada.

We are grateful to S. Turkarslan (University of Philadelphia) for providing plasmid pDsbBWT and for helping us with the construction of strains MD22/pDsbBWT and MT1131/pDsbBWT.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, General Microbiology Unit, Faculty of Sciences, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy. Phone: 39051 2091285. Fax: 39051 242576. E-mail: davide.zannoni{at}unibo.it. Back

{triangledown} Published ahead of print on 10 November 2006. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, February 2007, p. 851-859, Vol. 189, No. 3
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