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Applied and Environmental Microbiology, May 2006, p. 3147-3153, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3147-3153.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Genetic and Biochemical Evidence for the Involvement of a Molybdenum-Dependent Enzyme in One of the Selenite Reduction Pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Bénédicte Pierru, Sandrine Grosse, David Pignol, and Monique Sabaty^*

Laboratoire de Bioénergétique Cellulaire, CEA/Cadarache, DSV-DEVM, 13108 St. Paul lez Durance Cedex, France

Received 3 January 2006/ Accepted 16 February 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selenium (Se) is a naturally occurring element, essential for life at low concentrations as part of selenocysteine, a residue involved in the active site of various prokaryotic and eukaryotic enzymes (32). However, it can become toxic at high concentrations, as may happen due to its widespread use in industrial and agricultural processes. High selenium concentrations can cause important ecological problems, such as in the Kesterson reservoir in the San Joaquin Valley, where its accumulation led to severe abnormalities in the development of aquatic organisms (27). In aerobic environments, selenium predominantly occurs in the forms of selenate (SeO₄^2–) and selenite (SeO₃^2–) oxyanions. Both of these soluble inorganic oxidized forms, which are highly toxic, have been shown to bioaccumulate in the food chain (22). Several studies carried out with Salmonella enterica serovar Typhimurium (14), Rhodobacter sphaeroides (1), and Escherichia coli (2) have revealed the involvement of superoxide dismutase in response to selenium oxides, suggesting that the mechanism of toxicity of selenate and selenite could be related to oxidative damage.

Some microorganisms can, however, resist high concentrations of these oxyanions. Detoxification of these compounds can occur by volatization in the environment through methylation into dimethyl selenide and dimethyl diselenide. This process, catalyzed by bacterial thiopurine methyltransferases (21), has been detected in soil, sediment, and water (6). Detoxification can also be achieved by reduction of selenate and selenite into the elemental form Se⁰, which is insoluble and nontoxic. Depending upon the species, this process is generally followed by intracellular sequestration of the insoluble metallic form in the cytoplasm (11, 23), which is of great interest for bioremediation. Excretion of Se⁰ outside the cell has also been described (12, 41). In a few species of bacteria, selenate and selenite act as electron acceptors in an anaerobic form of respiration (16, 33, 34), whereas for most of the microorganisms studied so far, reduction of selenium oxyanions is not a bioenergetic process. Some species, including E. coli (36), Thauera selenatis (4, 29), and Enterobacter cloacae SLD1a-1 (15), have been reported to reduce both selenate and selenite; others, like R. sphaeroides (38) and Ralstonia metallidurans (28), can only reduce selenite.

Various putative mechanisms for selenium oxide reduction have been suggested. Since selenite is highly reactive with sulfhydryl groups, glutathione, which is one of the most abundant thiol in eukaryotic and prokaryotic cells, could be involved. Moreover, chemical approaches have revealed that selenite can react with glutathione in vitro, leading to the production of intermediate metabolites such as selenodiglutathione, glutathioselenol, hydrogen selenide (HSe^–), and finally elemental selenium (10). In addition to this chemical reduction, enzymatic processes have also been described. The reduction of oxyanions has often been associated with denitrification, since bacterial nitrate reductases have been shown to catalyze selenate reduction (24, 40). However, T. selenatis and E. cloacae SLD1a-1 possess a specific selenate reductase (4, 39). In both species, the enzymes belong to the molybdenum-dependent reductase (molybdoreductase) family. Only the enzyme from T. selenatis has been biochemically characterized. It is folded as a complex made of a catalytic molybdenum-containing subunit associated with a cytochrome b, and a binding subunit made up of two [Fe-S] clusters (29). In this species, selenite reduction may be achieved by the nitrite reductase (4). The involvement of a molybdenum enzyme in the reduction of selenate has also been suggested for E. coli, since mutants affected in the synthesis of molybdopterin are impaired in selenate reduction (2).

In the present study, we have combined biochemical and genetic strategies to characterize the reduction of selenite in the nonsulfur photosynthetic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Our results are consistent with the existence of several pathways of reduction, one of them involving an uncharacterized molybdoenzyme, different from nitrate, biotin sulfoxide, and dimethyl sulfoxide (DMSO) reductases.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial growth conditions.
Cultures of R. sphaeroides were grown at 30°C either in Sistrom minimal medium supplemented with succinate (31) or in Hutner medium (3) using Schott bottles sealed with rubber septa and degassed with a vacuum pump. For phototrophic growth, the light intensity was 75 µmol of photons · m^–2 · s^–1 (microeinstein · m^–2 · s^–1). When indicated, the medium was supplemented with 1 mM Na₂SeO₃, 1 mM Na₂WO₄, 20 mM KNO₃, or 80 mM DMSO. When appropriate, kanamycin was added at 25 µM.

Selenite concentration assays.
The residual concentration of selenite in the culture medium or in the buffer, when kinetics were measured, was determined according to Kessi et al. (11).

Growth curves.
Hutner medium supplemented with 1 mM selenite was inoculated with a culture in the exponential phase to an optical density at 660 nm (OD₆₆₀) of 0.1. Samples were taken at different times. The cell concentration was determined from the absorbance at 660 nm (OD₆₆₀). For OD₆₆₀ values of >0.6, culture samples were diluted before measurement. The samples were centrifuged, and the supernatant was frozen for subsequent selenite concentration measurement.

Kinetics of reduction.
Kinetics of selenite reduction were monitored by measuring the depletion of selenite over time according to Taratus et al. (35). The culture was grown phototrophically in Sistrom medium in the absence of selenite but in the presence of 20 mM nitrate or 80 mM DMSO when mentioned. In late exponential phase (OD₆₆₀ = 2), the culture was treated with chloramphenicol (150 µg · ml^–1) to inhibit protein synthesis, centrifuged (6,800 x g; 5 min), and resuspended to an OD₆₆₀ of 4 in 70 mM Na₂HPO₄, 30 mM NaH₂PO₄, 1 mM MgSO₄, 200 µM MnSO₄, and 30 mM lactate. Selenite (1 mM) was then added to 3 ml of cell suspension before incubation at 30°C under light. Samples were taken for analysis at different times and centrifuged, and the supernatant was frozen for subsequent selenite concentration measurement.

Library screening.
Mutants affected in selenite reduction were selected from a Tn5 transposon library (1) as follows: mutants were grown in 96-well microtitration plates in Hutner medium under photosynthetic anaerobic conditions. During the exponential growth phase, 1 mM selenite was added to the medium, and the appearance of a red color due to Se⁰ was monitored. Clones presenting a slower appearance of the red color were selected. Among them, the mutants affected in growth, even in the absence of selenite, were eliminated, since they all presented reduced selenite reduction activity due to their decreased growth.

Location of the Tn5 insertion was determined by an inverse PCR method as described by Bebien et al. (1). Sequence determination was performed by Genome Express (Grenoble, France).

Preparation of cell extracts and electrophoresis.
Preparation of cell extracts, nondenaturing gel electrophoresis, and activity staining were performed as previously described (25).

Strains and plasmid constructions.
For the DMSO reductase mutant, a 1-kb fragment of the dmsA gene was amplified from chromosomal DNA with the primers dmsA1719 (5'-CTACGAGCGCAACGACATCG-3') and RdmsA2719 (5'-AAGCGGGTGATGTCACAAGT-3'). The PCR product was cloned into pGEM-T Easy (Promega). An omega cartridge encoding resistance to streptomycin and spectinomycin (20) was then cloned into the BamHI site of dmsA. The resulting plasmid was digested with PstI and NcoI, and the fragment containing the dmsA-disrupted gene was cloned into pSUP202Km (30) restricted with the same enzymes. The resulting plasmid, unable to replicate into R. sphaeroides, was moved from E. coli to R. sphaeroides by conjugation. The double-crossover event was confirmed by Southern analysis and the disappearance of DMSO reductase activity.

Cloning bisC.
The DNA fragment containing the bisC gene was amplified from chromosomal DNA of R. sphaeroides f. sp. denitrificans by PCR with the primers Xba-BSO (5'-GCTCTAGAGCTAATCTGAAGGGGCGAGAGA-3') and rBSO6HEco (5'-CGGAATTCCGTCAGTGATGGTGATGGTGGTGAGTGGGCAGGATGGCTGCCA-3') to add a six-His tag to BisC. The PCR product was inserted into a pCR II-TOPO vector (Invitrogen). The resulting plasmid was digested with XbaI and EcoRI to obtain a 2.2-kb fragment containing bisC, which was cloned into XbaI- and EcoRI-restricted pBBR1MCS2 (13), yielding pMS718.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of selenite on the growth of R. sphaeroides f. sp. denitrificans.
It was established previously (1) that R. sphaeroides can grow aerobically or anaerobically on selenite-containing medium. During growth, the cells turned red as a result of selenite reduction into Se⁰ (17). When cultures were carried out in the presence of selenate, no reddening of the culture medium was observed, suggesting that R. sphaeroides lacked the ability to efficiently reduce Se(VI) to Se(IV) or to import selenate. The growth curves of R. sphaeroides cultivated in anaerobic conditions on Hutner medium in the presence or absence of 1 mM selenite are presented in Fig. 1. Selenite and/or Se⁰ accumulation was toxic for the bacteria, since a threefold decrease in the maximal cell density was observed in the presence of 1 mM selenite. Moreover, the addition of selenite blocked the onset of growth for about 100 h. The concentration of selenite in the medium was monitored as a function of time (Fig. 1). The disappearance of selenite was not due to its volatization through methylation into dimethyl selenide or dimethyl diselenide. Indeed, we verified by inducted coupled plasma atomic emission spectrometry that at the end of the experiment, the total amount of selenium species remaining in the culture was at least 95% of what was added at the start of the experiment (data not shown). Thus, the depletion of selenite was essentially due to its reduction into Se⁰. The maximal reduction rate of the oxyanion occurred during the exponential phase. The selenite concentration reached a value close to zero after 6 days of culture (Fig. 1). The same experiment was carried out after inoculation of fresh medium with a culture previously grown in the presence of 1 mM selenite (Fig. 1). In this case, the lag period was not observed further, selenite reduction occurred immediately, but the final cell density was still affected. This result reveals that the reduction of selenite involves an inducible pathway.

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FIG. 1. Growth curves of R. sphaeroides f. sp. denitrificans under anaerobic photosynthetic conditions in Hutner medium in the absence (•) or in the presence () of 1 mM selenite or in the presence of selenite after inoculation with a preculture grown in the presence of selenite (). Open symbols, depletion of selenite from the corresponding culture medium.

Characterization of mutants defective in selenite reduction.
To identify the genetic determinants of the selenite reduction, mutants affected by selenite reduction were selected from a transposon Tn5 library composed of 2,600 clones (1). We failed to obtain a mutant totally unable to reduce selenite from this library and other mutagenesis experiments (>15,000 mutants), and only mutants with decreased selenite reduction were characterized. The presence of a unique insertion was verified by Southern analysis. The identification of the disrupted genes was done by an inverse PCR method. The sequences of the regions surrounded the transposon were compared to the genome of R. sphaeroides 2.4.1 (http://mmg.uth.tmc.edu/sphaeroides/). In four mutants out of the eight selected from the screening, the transposon was inserted into regulatory genes like hipB (two mutants), hupT, or RSP 0169. In two mutants, the inactivated genes encoded a putative transporter or permease (smoM; RSP 1564). For only two mutants, the inactivated genes could be more easily related to a reduction pathway: mogA, a gene encoding a molybdochelatase responsible for the incorporation of molybdenum into molybdopterin, and moaA, a gene encoding a protein involved in the first step of the biosynthesis of an intermediate to the mature cofactor, designated precursor Z (for a review, see reference 9). A requirement for molybdocofactor (MoCo) synthesis in the selenite reduction pathway appeared possible, since molybdoenzymes have been shown in several instances to be related to selenate reduction (2, 29, 39). These two mutants were therefore studied further.

The genomic environment of moaA and mogA was determined by the partial sequencing of the surrounding genes, followed by a systematic comparison of the sequences with the Rhodobacter sphaeroides 2.4.1 genome (Fig. 2). In these regions, the two strains presented the same genetic organization. moeA, another gene involved in the molybdenum cofactor synthesis, is located 131 bp downstream of the dms operon, which encodes proteins necessary for DMSO reduction, and 57 bp upstream of moaA. At 179 bp downstream, moaA, a 2-kb open reading frame in the R. sphaeroides 2.4.1 genome designated RSP 3051, displayed weak similarities with putative alkaline phosphatases. The genome of R. sphaeroides 2.4.1 contained two copies of the moaA gene, sharing 74% identity. Southern experiments also revealed at least two copies of the moaA gene in R. sphaeroides f. sp. denitrificans (data not shown). Contrary to the findings with moaA, mogA was present as a single copy in the genome. mogA was surrounded by genes RSP 1824 and RSP 1822, transcribed in opposite directions. RSP 1824 encoded a putative alcohol dehydrogenase, and RSP1822 encoded a putative NAD⁺ synthetase.

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FIG. 2. Genetic organization around the moaA and mogA genes in Rhodobacter sphaeroides 2.4.1 (http://mmg.uth.tmc.edu/sphaeroides/). The location of the Tn5 insertion is indicated. The exact point of insertion is given as base pairs after the start of the coding sequence.

The phenotypes of these two mutants were further analyzed and compared to the wild-type strain. We first tested the synthesis of functional molybdoenzymes in these strains by monitoring the nitrate and DMSO reductase activities in periplasmic extracts (Fig. 3). As expected, the mogA mutant did not synthesize any more active nitrate or DMSO reductases (Fig. 3). On the other hand, the moaA mutant still synthesized these two molybdoguanine dinucleotide enzymes, although at a decreased level compared to that of the wild type (Fig. 3). This result is likely related to the presence of several copies of moaA in the genome, maintaining a correct maturation of part of the molydoreductases in the mutant.

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FIG. 3. Nondenaturing gel electrophoresis of periplasmic extracts of R. sphaeroides f. sp. denitrificans wild type (lane 1), moaA mutant (lane 2), and mogA mutant (lane 3). (A) Gels were stained for DMSO or nitrate reductase activity with reduced methylviologen as electron donor. (B) Gels were stained with Coomassie brilliant blue.

Cultures of the two mutants were performed in the absence or presence of 1 mM selenite in the medium. The lag period in the growth curve of the mutants was longer, 150 h instead of 100 h for the wild type (Fig. 4). Since both mutants were initially selected from a qualitative screening, we searched for a more accurate assay of selenite reduction. To obtain comparable results between the different strains, cultures of bacteria grown in the absence of selenite were harvested, treated with chloramphenicol, and resuspended in a phosphate buffer at the same cell density (OD₆₆₀ = 4). Thanks to the high concentration of cells, selenite reductase activity was measurable in these noninduced cultures (Fig. 5A), showing that selenite reductase is synthesized at low levels, even in the absence of selenite. It was not possible to use a similar procedure for cultures grown in the presence of selenite, because the bacteria were rendered very fragile due to the presence of Se granules and did not withstand the necessary centrifugation steps. The mogA mutant presented a significant decrease (by around 40%) in the reduction rate (Fig. 5B). When the same experiment was carried out with the moaA mutant, we obtained variable results, ranging from mogA mutant behavior to that of the wild type. This unstable phenotype may well be related to the existence of two copies of moaA gene and the partial synthesis of molybdoenzymes in this strain.

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FIG. 4. Growth curves of R. sphaeroides wild type (), moaA mutant (), and mogA mutant () in Hutner medium supplemented with 1 mM selenite, under photosynthetic conditions.

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FIG. 5. (A) Kinetics of selenite reduction in R. sphaeroides f. sp. denitrificans wild type or mogA mutant. Bacteria were resuspended in phosphate buffer (OD660 = 4) supplemented with 1 mM selenite. (B) Selenite reduction yields over 2 h of the wild-type culture cultivated in Hutner medium in the presence of 1 mM tungstate or of the mogA mutant (in the absence of tungstate) are presented in comparison to the wild-type culture in nonsupplemented medium, considered 100% (which represents 0.4 mM selenite reduced per hour for a culture with OD660 = 4). The results are the averages of at least three independent experiments.

Effect of tungstate on selenite reduction.
To gain further evidence for the involvement of a molybdoenzyme in the reduction of selenite, wild-type cultures were grown in the presence of sodium tungstate (1 mM), which is known to inactivate numerous molybdoenzymes through the replacement of W by Mo at their active site (7). We first verified that addition of tungstate in the culture had no effect on the growth in the absence of selenite (Fig. 6). Nevertheless, in the presence of selenite, the addition of tungstate led to a 50-h increase in the lag phase (Fig. 6). Moreover, the reduction rate of selenite by cells grown in the presence of tungstate was decreased by around 40% (Fig. 5B). In all respects, the effect of tungsten mimicked results obtained with the mogA mutant.

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FIG. 6. Growth curves of R. sphaeroides f. sp. denitrificans under anaerobic photosynthetic conditions in Hutner medium with no addition (•), 1 mM tungstate (), 1 mM selenite (), and 1 mM tungstate plus 1 mM selenite (). Open symbols, depletion of selenite from the corresponding culture media.

Taken together, these results suggest that a molybdoenzyme is involved in one selenite reduction pathway, which may represent 40% of the total selenite reduction activity. We therefore searched for possible candidates among known molybdoenzymes.

Role of nitrate, DMSO, and biotin sulfoxide reductases.
To test the putative role of DMSO and nitrate reductases in selenite reduction, we first studied the effects of the addition of nitrate and DMSO in the culture medium, since the synthesis of these molybdoenzymes is known to be induced by their respective substrates (26, 37). Cells were first grown in medium supplemented with nitrate or DMSO, and their ability to reduce selenite was monitored. The addition of nitrate or DMSO during growth markedly increased reduction activity by 3.5-fold and 2.1-fold, respectively (Fig. 7). This result could be explained by the ability of DMSO and nitrate reductases to catalyze selenite reduction. To test this hypothesis, we focused on two mutants, napA (26) and dmsA (this study), with expression of periplasmic nitrate reductase and DMSO reductase, respectively, deleted. Both of them reduced selenite at the same rate as the wild type (data not shown). Moreover, the inducing effect of nitrate was also observed, to a lesser extent, with the napA mutant (Fig. 7). As it has been established that the periplasmic nitrate reductase Nap is the only active nitrate reductase in this strain (26), these results show that nitrate and DMSO reductases are not the molybdoenzymes involved in this reduction pathway.

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FIG. 7. Selenite reduction activity of R. sphaeroides f. sp. denitrificans wild type or napA mutant grown under photosynthetic conditions in Sistrom medium, in the absence or presence of 20 mM nitrate or 80 mM DMSO. Bacteria were resuspended in phosphate buffer (OD660 = 4) supplemented with 1 mM selenite. Selenite reduction yields over 2 h are presented in comparison to the wild-type culture in nonsupplemented medium, considered as 100% (which represents 0.12 mM selenite reduced per hour for a culture of OD660 = 4).

Biotine sulfoxide reductase BisC (19) was another candidate that could play a role in selenite reduction, since this molybdoenzyme has been shown to exhibit a wide substrate specificity in vitro, including biotin sulfoxide, DMSO, trimethylamine oxide, and methionine-S-sulfoxide (5, 18). In addition, the corresponding gene, bisC, was not present in Rhodobacter sphaeroides 2.4.1, a strain exhibiting a very low level of selenite reduction activity (10-fold lower than R. sphaeroides f. sp. denitrificans) (data not shown). We therefore tried to express BisC from R. sphaeroides f. sp. denitrificans in R. sphaeroides 2.4.1. bisC (His-tagged) was cloned into pBBR1MCS2, and the resulting plasmid (pMS718) was introduced into R. sphaeroides 2.4.1 and R. sphaeroides f. sp. denitrificans by conjugation. We verified that BisC was overexpressed by using Western blots with antibodies against the His tag (data not shown). The selenite reductase activities of R. sphaeroides 2.4.1 exhibiting the empty plasmid or the plasmid harboring bisC were monitored. No difference was observed, however, between the two strains (data not shown). These results would suggest that BisC is not the molybdoenzyme involved in selenite reduction.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many microorganisms have been reported to resist high concentrations of selenite but with different behaviors. For some, like R. metallidurans (23), the final cell density is not affected by the presence of selenite. In contrast, as observed for Rhodospirillum rubrum (11), we showed that growth of R. sphaeroides f. sp. denitrificans cultivated under photosynthetic conditions in Hutner medium was affected, since the addition of 1 mM selenite decreased the final optical density by a factor of 3 (Fig. 1). Bebien et al. (1) did not observe this effect. However, in the absence of selenite, their cultures only reached an OD₆₆₀ of 3.2, while we obtained an OD₆₆₀ of 6.7, suggesting a major difference in the medium composition or in the culture conditions. Nevertheless, in both studies, the selenite consumption took place during the exponential phase for R. sphaeroides, whereas it occurred during the transition from late exponential to stationary phase for R. rubrum and for R. metallidurans (11, 23). The localization of the Se⁰ deposits is another feature that differs among resistant strains (11, 12, 23). Different mechanisms have been reported for the detoxification of selenite by bacteria. One of them is the volatilization through methylation into dimethyl selenide and dimethyl diselenide by bacterial thiopurine methyltransferases (6, 21). Reduction to the elemental form Se⁰ has also been widely described. It has been suggested that selenite can react with the intracellular pool of glutathione. Indeed, in vitro experiments have shown that abiotic reduction of selenite into Se⁰ occurs in the presence of glutathione with selenodiglutathione as an intermediate (10). In addition to this chemical reduction, an enzymatic process has also been reported for T. selenatis or Clostridium pasteurianum, where selenite reduction is achieved by nitrite reductase (4) or hydrogenase I (42), respectively.

Our results bring evidence that an enzymatic pathway requiring MoCo synthesis is operative in R. sphaeroides f. sp. denitrificans and that this is not the sole mechanism involved. A first indication for the involvement of an enzymatic process is the 100-h lag phase preceding the onset of rapid selenite reduction, which is suppressed when the medium is inoculated with a culture previously grown in the presence of selenite (Fig. 1). This is suggestive of an inducible enzymatic mechanism. The inactivation of genes involved in the synthesis of the molybdenum cofactor or the addition of tungstate (an inhibitor of molybdoenzymes) in the culture medium decreased the selenite reduction turnover (Fig. 5B) and increased the duration of the lag phase (Fig. 4 and 6). The inactivation of the molybdenum cofactor caused only a partial suppression of selenite reduction activity. Both the inactivation of mogA or growth of the wild type in the presence of tungstate resulted in only a 40% decrease in the constitutive rate of selenite reduction. Similarly, when the mogA mutant was cultivated in the presence of selenite, cell growth and selenite reduction were slowed down with respect to the wild type but not suppressed. It is thus clear that another reduction pathway, involving no MoCo, is operative. The coexistence of several pathways actually appears very likely, considering the impossibility of isolating a mutant of R. sphaeroides (out of 15,000 isolated mutants of R. sphaeroides) totally impaired in selenite reduction, a result also found with R. metallidurans and Shewanella oneidensis (J. Coves and A. Klonowska, personal communication).

Several possibilities can be considered concerning the MoCo-independent pathway. We can exclude the methylation of selenite to volatile compounds, since we showed that >95% of the Se remains present in the culture. With T. selenatis, it has been reported that selenite reduction could be catalyzed by nitrite reductase (4), which is not a molybdoenzyme. We tested this possibility by assaying activity by nondenaturing gel electrophoresis, using methyl or benzyl viologen as electron donors. We previously showed in this manner that selenate reduction could be achieved by the periplasmic nitrate reductases of several strains (24). However, we could not detect any selenite reductase activity under the same conditions, even when nitrite reductase activity was clearly present (data not shown). It thus appears unlikely that the nitrite reductase of R. sphaeroides is responsible for the MoCo-independent reduction of selenite. We feel that chemical reduction by glutathione or other reducing compounds is the likeliest possibility, although the occurrence of a second selenite reductase devoid of MoCo cannot be excluded.

We investigated several candidates among the known molybdoenzymes that might account for MoCo-dependent selenite reductase activity. Nap, DMSO reductase, and BisC appeared to be the most likely candidates. Although the presence of nitrate markedly increased the ability of the bacteria to reduce selenite, it turned out that Nap was not responsible for this activity, since the selenite reduction activity is identical for the wild type and the nap mutant (Fig. 7). Contrary to other strains, which possess both membrane-bound and periplasmic nitrate reductases, the only active nitrate reductase in R. sphaeroides f. sp. denitrificans is Nap (26). Thus, selenite reduction in this strain is not due to a nitrate reductase. The same conclusion can be drawn for DMSO reductase, based on the phenotype of the dmsA mutant. We suggest that the induction effect caused by nitrate or DMSO is related to the activation of the synthesis of the molybdenum cofactor rather than the apoenzymes. Indeed, in Escherichia coli, the transcription of moeA, encoding an enzyme involved in molybdenum cofactor synthesis, is regulated by nitrate and trimethylamine N-oxide, since moeA-lacZ expression increases 3.5- and 1.5-fold, respectively, in the presence of these substrates (8). A direct effect of DMSO and nitrate on the selenite reductase synthesis seems improbable but cannot be excluded. Finally, a role for BisC, another molybdoenzyme whose function in the cell is not well established, seems unlikely. Indeed, its overexpression in R. sphaeroides 2.4.1, which exhibits a selenite reduction activity 10-fold lower than that of R. sphaeroides f. sp. denitrificans, does not affect this activity. However, one cannot totally exclude that the poor reduction activity of R. sphaeroides 2.4.1 harboring bisC could be due to a less efficient import system for selenite.

The molybdoenzyme responsible for the selenite reduction activity in R. sphaeroides f. sp. deniftrificans is thus still not identified.

 
This work was supported by the Commissariat à l'Energie Atomique (CEA). B.P. is a doctoral fellow of the Programme de Toxicologie Nucléaire Environnementale.

We thank Jérôme Lavergne and Pascal Arnoux for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Laboratoire de Bioénergétique Cellulaire, CEA/Cadarache, DSV-DEVM, 13108 St. Paul lez Durance Cedex, France. Phone: 33 4 42 25 35 70. Fax: 33 4 42 25 47 01. E-mail: msabaty{at}cea.fr.

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Applied and Environmental Microbiology, May 2006, p. 3147-3153, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3147-3153.2006
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