CEA/Cadarache-DSV-DEVM-Laboratoire de Bioénergétique Cellulaire, Université de la Méditerranée CEA 1000, 13108 Saint-Paul-lez-Durance Cedex, France1
Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, 31 Chemin Joseph Aiguier, BP 71, 13402 Marseille Cedex 20, France2
Author for correspondence: André Verméglio. Tel: +33 442254630. Fax: +33 442254701. e-mail: avermeglio{at}cea.fr
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ABSTRACT |
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Keywords: molybdenum ironsulfur protein, oxyanion reduction, selenite
Abbreviations: EDX, energy-dispersive X-ray; TMAO, trimethylamine N-oxide
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INTRODUCTION |
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In the biogeochemical cycle of selenium, various redox reactions are carried out by microorganisms. Several bacteria, including Escherichia coli (Turner et al., 1998 ) are able to reduce both selenate and selenite into elemental selenium (Se0), while certain species like Rhodobacter sphaeroides (Bebien et al., 2001
; Van Fleet-Stalder et al., 2000
) or Ralstonia metallidurans (Roux et al., 2001
) reduce only selenite. The reduction of the bioavailable selenium oxyanions into elemental selenium, which is insoluble and non-toxic, is of great interest for bioremediation. Particles of elemental selenium accumulate in the periplasm (Gerrard et al., 1974
), in the cytoplasm (Silverberg et al., 1976
) or outside the cell (Yamada et al., 1997
), depending upon the species. The biological mechanisms and enzymes involved in the reduction of selenate and selenite are still to be characterized. Up to now, a specific selenate reductase has only been purified from Thauera selenatis (Schröder et al., 1997
), a species that uses selenate or selenite as electron acceptors in the first steps of an anaerobic respiratory process similar to denitrification (DeMoll-Decker & Macy, 1993
). This highly specific reductase does not reduce nitrate, nitrite, chlorate or sulfate. The enzyme has an apparent molecular mass of 180 kDa and is composed of three subunits. It contains a cytochrome b, a molybdenum cofactor and two putative [FeS] centres. In addition, the second step of selenate reduction to selenium the reduction of selenite to Se0 is catalysed by the nitrite reductase in T. selenatis. This has been clearly demonstrated by the inability of a mutant defective in this enzyme to sustain selenite reduction (DeMoll-Decker & Macy, 1993
). Other evidence for the involvement of the denitrification enzymes in selenate or selenite reduction comes from the observation that in situ nitrate and selenate reductions present similar profiles as a function of the sediment depth (Oremland, 1994
). Moreover, in vitro studies have shown that soluble or membranous nitrate reductases of different species possess selenate reductase activity (Avazeri et al., 1997
). Extensive in vitro studies on the mechanisms of selenite reduction have shown the involvement of thiol groups of thiol-containing molecules such as glutathione (GSH) leading to the production of the intermediate metabolites selenodiglutathione (GSSeSG), glutathioselenol (GSSeH) and hydrogen selenide (HSe-) and finally to elemental selenium (Ganther, 1968
; Kice et al., 1980
). One consequence of these reactions is the production of the highly toxic hydrogen peroxide (H2O2) and superoxide (
) suspected of causing damage to the cell membranes and DNA (Kramer & Ames, 1988
; Seko & Imura, 1997
). Recently, we have described the in vivo enhancement of the synthesis of enzymes associated with oxidative stress in response to selenate or selenite addition in both E. coli (Bebien et al., 2002
) and the photosynthetic bacterium Rb. sphaeroides (Bebien et al., 2001
). To prevent acute and chronic toxicity of the soluble selenium compounds, it is important to establish the molecular and physiological basis of the detoxification and removal of selenium oxyanions.
In the present work, molecular genetic strategies have been used to investigate the mechanisms responsible for the reduction of selenium oxyanions, in particular selenate, in E. coli.
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METHODS |
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Screening procedure to isolate mutants defective in selenate reduction and DNA manipulation.
The random mini-Tn10 transposition mutagenesis was described by Ansaldi et al. (1999) . Tn10 insertion mutants were grown on LB plates containing chloramphenicol (12·5 µg ml-1) and incubated for 23 days at 37 °C. Mutants were subsequently plated on LB medium containing
(1 mM) to screen the mini-Tn10 insertions for lack of selenate reduction ability. The location of the insertions was analysed using a rapid inverse PCR method (Ansaldi et al., 1999
). The PCR product was purified from an agarose gel using a PCR purification kit (QIAquick, QIAgen). DNA sequencing was performed with an ABI apparatus (ABI Prism 310, Applied Biosystems). DNA and protein sequence analyses were performed using software tools (ExPASy).
Minimal inhibitory concentration (MIC) determination.
Determination of the MIC, defined as the lowest concentration of inhibitor preventing growth of E. coli strains at 37 °C on agar plates, was performed as described previously (Avazeri et al., 1997 ).
Electron microscopy and X-ray analysis.
Cells were fixed in 2·5% glutaraldehyde and 0·1 M cacodylate buffer, pH 7·1, for 30 min. After washing twice with the same medium, they were post-fixed in 1% OsO4 in 0·02 M cacodylate buffer, pH 7·1, for 1 h, and subsequently dehydrated with a graded ethanol/water series and embedded in low-viscosity epoxy resin (Epon). Microtome-cut thin sections were contrasted with uranyl acetate and lead citrate, as described by Hess (1966) , and observed with a Philips CM 120 transmission electron microscope. For energy-dispersive X-ray (EDX) analysis, thin sections were applied to carbon-coated transmission electron microscopy grids and dried at room temperature. The EDX analysis was performed with a JEOL model 2010 F electron microscope operating at 200 kV and equipped with an EDAX-KEVEX microanalysis system.
Chemicals.
Sodium selenate and sodium selenite were purchased from Sigma-Aldrich. The oligonucleotides used were purchased from Genome express. All other chemicals used were analytical grade.
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RESULTS AND DISCUSSION |
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Transport of selenate
One mutant that is unable to reduce selenate is affected in the sulfate uptake ATP-binding protein (CysA). This result was not surprising, since previous studies have clearly demonstrated that selenate enters the cell through this system in E. coli (Linblow-Kull et al., 1985 ) and Saccharomyces cerevisiae (Smith et al., 1995
). In addition, we found that mini-Tn10 insertions in ybaT or nmpC genes, encoding a putative amino acid or metabolite transport protein probably located in the inner membrane and an outer-membrane porin protein (Table 2
), respectively, inhibited the reduction of selenate without any change to the growth rate. This implies that, in addition to the sulfate permease system, these two proteins are essential for the transport of selenate from the outside of the bacteria into their cytoplasm.
Requirement of a molybdenum-containing enzyme for selenate reductase activity
The mutants moeA and molR are both defective in genes encoding enzymes required for biosynthesis of the molybdenum cofactor, which consists in the most simple form of a pterin complexed to molybdenum (Table 2). This strongly suggests that the enzyme involved in the reduction of selenate into selenite is a molybdenum-containing enzyme. To test this hypothesis further, we measured the selenate-reducing capability of a series of mutants deleted in genes involved in the synthesis of the molybdopterin (MPT) cofactor. The two mutants RK5200 and RK5202 (Table 1
), deleted in moa and mob operons (formerly designated chlA and chlB), responsible for biosynthesis of the mononucleotide and dinucleotide forms of the pterin cofactor (reviewed by Hille, 1996
), respectively, were unable to reduce selenate. In addition, selenate reduction was not observed for the mutant RK5208 defective in mod locus (chlD in the former nomenclature), which encodes a high-affinity molybdate uptake system. However, in agreement with previous reports, showing that molybdate can enter the cell by other pathways (Rosentel et al., 1995
), the addition of high concentrations of molybdate to the growth medium restored molybdoenzyme activities such as nitrate reductase activity (Rosentel et al., 1995
; this work) and, consequently, the capability to reduce selenate. These results demonstrate the involvement of a molybdoenzyme in the reduction of selenate to selenite.
E. coli contains several molybdopterin guanine dinucleotide (MGD)-dependent enzymes including the arsenate, DMSO, TMAO and nitrate reductases and the formate dehydrogenases. Particular attention was paid to the possible role of the different nitrate reductases in the selenate reduction. Indeed, previous in vitro studies have demonstrated that the membrane-associated nitrate reductases of E. coli (NRA, NRZ) were able to reduce selenate with benzyl viologen as electron donor (Avazeri et al., 1997 ). More recently, this phenomenon was described to be a general feature of various soluble or membrane-bound nitrate reductases of numerous denitrifying species (Sabaty et al., 2001
). However, a mutant of E. coli deleted in both membrane-bound reductases NRA and NRZ (LCB2048) still reduces selenate, resulting in the accumulation of elemental selenium. In addition to the two membrane-bound nitrate reductases NRA and NRZ, E. coli harbours a dissimilatory periplasmic nitrate reductase (Nap system, reviewed by Moreno-Vivian et al., 1999
). The role of this soluble enzyme in the reduction of selenate was tested using a strain lacking both nar and nap genes (JCB20480). This triple mutant turned red, like the wild-type, in the presence of selenate, resulting from the reduction of this oxyanion into metallic selenium. Furthermore, the MIC of selenate is identical for the wild-type and the
nar
nap or
nar mutants.
In addition to nitrate reductases, we have observed that DMSO and TMAO reductases also possess a selenate reductase activity in vitro with benzyl viologen as electron donor (data not shown). This in vitro selenate reductase activity, however, is 10-fold lower than the selenate reductase activity of nitrate reductases. Mutants altered in DMSO, TMAO or arsenate reductases, or in formate dehydrogenases, were still able to reduce selenate into metallic selenium with intracytoplasmic accumulation (data not shown). We therefore conclude that, although some of the molybdoenzymes of E. coli possess selenate reductase activity in vitro, their contribution to the in vivo reduction of selenate is low or nil.
This series of results provides evidence that the reduction of selenate in E. coli is catalysed, as demonstrated for T. selenatis (Schröder et al., 1997 ), by a molybdoenzyme. In addition, this enzyme differs from various mononuclear oxomolybdenum enzymes described so far in E. coli.
Identification of a putative selenate reductase in E. coli
One of the mutants unable to reduce selenate into selenite was altered in the synthesis of a putative oxido-reduction enzyme, denoted YgfK. The alignment of the ygfK sequence in the databases shows some homology with the Pyrococcus abyssi glutamate synthase (36%) and the Clostridium thermoaceticum formate dehydrogenase (41%). Structurally, this enzyme of about 115 kDa consists of at least three distinct domains: an N-terminal NAD-binding domain, a central pyridine nucleotidedisulfide redox domain and a C-terminal ironsulfur binding domain (Fig. 2A). The NAD-binding domain has been found in a wide range of redox proteins, including alcohol dehydrogenases, amine oxidases, glutamate and other dehydrogenases. These enzymes have at least one NAD as redox cofactor that functions as an electron carrier in oxidationreduction processes. The pyridine nucleotidedisulfide domain is actually a small NADH-binding domain within a larger FAD-binding domain present in both class I and class II disulfide oxidoreductases. These enzymes are FAD flavoproteins such as the mercuric reductase of Bacillus RC607 (Schiering et al., 1991
), which contains a pair of redox-active cysteines involved in the transfer of reducing equivalents from the FAD cofactor to the substrate. The 4Fe4S centre of YgfK is similar to those of bacterial ferredoxins, various dehydrogenases and reductases, which mediate electron transfer in a wide variety of metabolic reactions. However, the complete analysis of the domain structure of YgfK does not show any similarity to known molybdopterin-binding domains. Thus, the focus was on genes present in the region of ygfK (Fig. 2B
). A putative operon, located downstream of ygfK, contains the genes ygfM and ygfN, which encode a 28·5 kDa and 104 kDa protein, respectively. The YgfM polypeptide shows a FAD-binding domain found in molybdopterin dehydrogenase. The protein YgfN contains a N-terminal [2Fe2S]-binding domain and a C-terminal molybdopterin-binding domain signature. This last domain is largely described in members of the xanthine oxidase family (Ald_Xan_Dh_C2). However, a comparison of the ygfN sequence in the databases shows only a low similarity to known proteins, limited to xanthine dehydrogenase (e.g. 25% from Bacillus halodurans) and aldehyde oxidoreductase (e.g. 28% from Desulfovibrio gigas). In addition, these three proteins YgfK, YgfM and YgfN are predicted to be soluble proteins without any leader peptides in their N-terminus. These proteins might form a structural complex involved in the reduction of selenate. This proposal takes into account our finding that the selenate reductase in E. coli requires a molybdopterin cofactor and is located in the cytoplasm. This proposal is also in line with the demonstration that the selenate reductase of T. selenatis is composed of several polypeptides encoded by the serABDC loci (Krafft et al., 2000
). Several attempts were made to provide biochemical evidence that YgfKMN is a selenate-reducing complex. Comparison of selenate-reduction activities of the soluble fractions of the wild-type and the ygfK mutant was not conclusive because of the high selenate reductase activity of the nitrate reductases present in these fractions. Another approach was to measure selenate reductase activities on non-denaturating gels with benzyl viologen as electron donor for the triple mutant lacking both nar and nap genes. However, this approach was not conclusive either, since no selenate activity could be detected in this mutant although it still readily reduced selenate in vivo. A definitive proof of the selenate reductive capacity of the YgfKYgfMYgfN complex will therefore necessitate the characterization of mutants deleted in ygfM and ygfN genes and a complete in vitro functional characterization of this complex after overexpression and purification.
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Concerning the reduction of selenite, we observed that all the different mutants affected either in the synthesis of the molybdenum cofactor or in a specific molybdoenzyme are still able to reduce this compound into elemental selenium. This implies that the different steps in the reduction of selenite into elemental selenium do not involve a molybdoenzyme. In addition, intracellular accumulation of metallic selenium was not impaired in a mutant of E. coli defective in the periplasmic nitrite reductase (JCB387). Since it has been clearly shown that the soluble nitrite reductase catalyses the reduction of selenite in T. selenatis (DeMoll-Decker & Macy, 1993 ), the presence of a different pathway highlights the various mechanisms developed by bacteria to reduce selenium oxyanions.
Conclusion
The present analysis of mutants impaired in selenate reduction selected from a random mini-Tn10 insertion library provides new insights into the mechanisms of transport and reduction of this oxyanion in E. coli. We first confirmed that selenate enters the cell through the sulfate permease, in agreement with the similarities between the chemical properties of sulfur and Se. In addition, we showed that selenate uptake into the cells also requires the transport protein YbaT and a functional outer-membrane porin. Our results indicated that selenate reductase in E. coli is a molybdoenzyme that differs from the various molybdoreductases described so far in this species. We propose that YgfK, YgfM and YgfN are three subunits of the selenate reductase of E. coli.
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ACKNOWLEDGEMENTS |
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Received 18 April 2002;
revised 22 July 2002;
accepted 27 August 2002.
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