A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter

The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300–0303 and Cj1538–1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate ( K _D values for both ligands of 4–8 nM), Cj1540 binds tungstate with a K _D of 1.0 ± 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 ( tupA ) mutant compared with a cj0303c ( modA ) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased ∼50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.     

Efficient bioaccumulation of tungsten by Escherichia coli cells expressing the Sulfitobacter dubius TupBCA system

Tungsten (W) is a valuable element with considerable industrial and economic importance that belongs to the European Union list of critical metals with a high supply risk. Therefore, the development of effective and new methods for W recovery is essential to ensure a sustainable supply.In the present study, the Sulfitobacter dubius W transport system TupABC was explored in order to demonstrate both its functionality in Escherichia coli cells and to construct a bioaccumulator (EcotupW). The complete gene cluster tupBCA or partial gene cluster tupBC were cloned in an expression vector and transformed into E. coli. Metal accumulation was evaluated when each construct strain was grown with three separate metal oxyanions (tungstate, molybdate or chromate). The specificity of the bioaccumulator was determined by competition assays using cells grown with mixed solutions of metal oxyanions (W/Mo and W/Cr). The results showed the relevance of the TupA protein in the TupABC transporter system to W-uptake and also allowed Mo and Cr accumulations, although with amounts 1.7 and 2.9-fold lower than W, respectively. To identify the importance of the valine residue in the accumulation efficiency of the VTTS motif, site-directed mutagenesis of tupA was performed. A mutant with a threonine residue, instead of the respective valine, confirmed that W was internalized by nearly double the amount compared to the native form.The findings indicated that cells carrying the native S. dubius TupABC system were great W-bioaccumulators and could be promising tools for W recovery.     

Tungstate Uptake by a highly specific ABC transporter in Eubacterium acidaminophilum

The Gram-positive anaerobe Eubacterium acidaminophilum contains at least two tungsten-dependent enzymes: viologen-dependent formate dehydrogenase and aldehyde dehydrogenase. (185)W-Labeled tungstate was taken up by this organism with a maximum rate of 0.53 pmol min(-)1 mg(-)1 of protein at 36 degrees C. The uptake was not affected by equimolar amounts of molybdate. The genes tupABC coding for an ABC transporter specific for tungstate were cloned in the downstream region of genes encoding a tungsten-containing formate dehydrogenase. The substrate-binding protein, TupA, of this putative transporter was overexpressed in Escherichia coli, and its binding properties toward oxyanions were determined by a native polyacrylamide gel retardation assay. Only tungstate induced a shift of TupA mobility, suggesting that only this anion was specifically bound by TupA. If molybdate and sulfate were added in high molar excess (>1000-fold), they were also slightly bound by TupA. The K(d) value for tungstate was determined to be 0.5 microm. The genes encoding the tungstate-specific ABC transporter exhibited highest similarities to putative transporters from Methanobacterium thermoautotrophicum, Haloferax volcanii, Vibrio cholerae, and Campylobacter jejuni. These five transporters represent a separate phylogenetic group of oxyanion ABC transporters as evident from analysis of the deduced amino acid sequences of the binding proteins. Downstream of the tupABC genes, the genes moeA, moeA-1, moaA, and a truncated moaC have been identified by sequence comparison of the deduced amino acid sequences. They should participate in the biosynthesis of the pterin cofactor that is present in molybdenum- and tungsten-containing enzymes except nitrogenase.     

Biosorption of tungstate by a Bacillus sp. isolated from Anzali lagoon

An attempt was made to isolate bacterial strains capable of biologically removing tungstate (WO₄²⁻). Thirty-eight water samples were collected from various areas of Anzali lagoon, Iran. Initial screening of a total of 100 bacterial isolates at pH 5, resulted in the selection of one isolate with maximum adsorption capacity of 65.4 mg tungstate/g dry weight. It was tentatively identified as Bacillus sp. according to morphological and biochemical properties and named strain MGG-83. Tungsten concentration was measured spectrophotometrically using the dithiol method. Higher adsorption capacity was observed in the acidic pH ranging from 1 to 3. At pH 2, the strain removed 274.4 mg tungstate/g dry weight within 5 min from the solution with 300 mg WO₄²⁻/l initial concentration and thereafter adsorption rate decreased remarkably. The applicability of the Freundlich isotherm for representation of the experimental data was investigated. Using 1 mM sodium azide and 10 mM 2,4−dinitrophenol, it was shown that only 20% reduction occurred in adsorption and steam sterilization of the bacterial cells resulted in 11% decrease in tungstate uptake. Temperature variations (20–40°C) had no significant effect on tungstate uptake. Pretreatment with the cations had no effect in uptake but pretreatment with anions decreased the tungstate uptake as indicated: sulfate > chromate > nitrate > molybdate > selenate > rhenate. Tungstate was removed from metal-laden biomass after desorption treatments by addition of different desorbing solutions with the results sodium acetate > EDTA > NaCl > KOH > H₂SO₄.     

Selenate Reduction to Elemental Selenium by Anaerobic Bacteria in Sediments and Culture: Biogeochemical Significance of a Novel, Sulfate-Independent Respiration

Interstitial water profiles of SeO₄²⁻, SeO₃²⁻, SO₄²⁻, and Cl⁻ in anoxic sediments indicated removal of the seleno-oxyanions by a near-surface process unrelated to sulfate reduction. In sediment slurry experiments, a complete reductive removal of SeO₄²⁻ occurred under anaerobic conditions, was more rapid with H₂ or acetate, and was inhibited by O₂, NO₃⁻, MnO₂, or autoclaving but not by SO₄²⁻ or FeOOH. Oxidation of acetate in sediments could be coupled to selenate but not to molybdate. Reduction of selenate to elemental selenium was determined to be the mechanism for loss from solution. Selenate reduction was inhibited by tungstate and chromate but not by molybdate. A small quantity of the elemental selenium precipitated into sediments from solution could be resolublized by oxidation with either nitrate or FeOOH, but not with MnO₂. A bacterium isolated from estuarine sediments demonstrated selenate-dependent growth on acetate, forming elemental selenium and carbon dioxide as respiratory end products. These results indicate that dissimilatory selenate reduction to elemental selenium is the major sink for selenium oxyanions in anoxic sediments. In addition, they suggest application as a treatment process for removing selenium oxyanions from wastewaters and also offer an explanation for the presence of selenite in oxic waters.     

Effect of Salinity on the Tolerance to Toxic Metals and Oxyanions in Native Moderately Halophilic Spore-forming Bacilli

Summary Ten moderately halophilic spore-forming bacilli were isolated from saline soils in Iran and their intrinsic high-level resistance to chromate, arsenate, tellurite, selenite, selenate and biselenite was identified by an agar dilution method. Minimum inhibitory concentration (MIC) for each oxyanion was determined. All isolates were resistant to higher concentrations of arsenate. The resistance level of the isolates to selenooxyanions was between 10 and 40 mM. Maximum and minimum tolerance against oxyanions was seen in selenite and biselenite, respectively. Although toxic metal resistance in the isolates was not different from non-halophilic bacteria that has been reported, unusual resistance to arsenate (250 mM), sodium chromate (75 mM) and potassium chromate (70 mM) was observed. The results obtained in this study revealed that all isolates were obviously susceptible to silver, nickel, zinc and cobalt, while seven isolates were resistant to lead. Susceptibility to copper and cadmium varied among the isolates. Silver had the maximum toxicity, whereas lead and copper showed minimum toxicity. The impact of salinity on the toxicity of oxyanions was also studied. Our results showed that in general an increase in salinity from 5% (w/v) to 15% (w/v) enhanced tolerance to toxic oxyanions.     

A Rhodobacter capsulatus Member of a Universal Permease Family Imports Molybdate and Other Oxyanions

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.Molybdenum (Mo) is utilized by many bacteria, archaea, and eukaryotes as a cofactor of redox enzymes catalyzing key reactions in the nitrogen, sulfur, and carbon cycles (62). Nitrogenase, which catalyzes the reduction of dinitrogen to ammonia, carries the unique iron-molybdenum cofactor FeMoco. In contrast to nitrogenase, all other molybdoenzymes harbor the molybdenum cofactor Moco, which transfers either an oxo group or two electrons to or from the substrate in a wide variety of transformations at nitrogen, sulfur, and carbon atoms (47).The phototrophic alphaproteobacterium Rhodobacter capsulatus serves as a model organism to study Mo metabolism because it synthesizes several molybdoenzymes, including dimethyl sulfoxide reductase, xanthine dehydrogenase, and nitrogenase (29, 30, 46). In addition to Mo-dependent nitrogenase (Mo-nitrogenase), R. capsulatus uses an alternative, Mo-free nitrogenase when Mo is limiting (55, 57). Two related Mo-responsive regulators, MopA and MopB, control expression of the alternative nitrogenase and molybdate uptake genes (22, 57, 58).Mo is available for living cells in its oxyanion form, molybdate. The vast majority of Mo-utilizing bacteria is known or predicted to possess ModABC-type high-affinity molybdate uptake systems (62, 63). These importers belong to the family of ATP-binding cassette (ABC) transporters, which couple ATP hydrolysis to substrate translocation across biological membranes (13, 15). ModABC transporters typically consist of a periplasmic molybdate-binding protein (ModA), a membrane-spanning channel protein (ModB), and a cytoplasmic ATP-binding protein (ModC), which specifically interacts with ModB and, upon ATP hydrolysis, energizes the uptake system.ModABC transporters enable bacteria to actively take up molybdate against a concentration gradient and synthesize active molybdoenzymes at nanomolar Mo concentrations in the environment (37, 49). Accordingly, modABC mutants are not able to make use of molybdoenzymes under Mo-limiting conditions, as shown for several bacteria, including Escherichia coli, Anabaena variabilis, Azotobacter vinelandii, and R. capsulatus (16, 33, 55, 61). High Mo concentrations, however, support synthesis of active molybdoenzymes in modABC mutants, indicating the presence of low-affinity molybdate uptake systems in these bacteria. Low-affinity molybdate uptake in E. coli and several other bacteria is mediated (at least in part) by the sulfate-repressed high-affinity sulfate transporter CysTWA (37, 43, 61).In the present study, we describe the identification and characterization of a permease mediating molybdate uptake at micromolar concentrations in R. capsulatus. The permease belongs to the widely distributed family of ArsB/NhaD permeases (27). Several members of this family have been shown to transport various anorganic and organic anions across biological membranes, but molybdate uptake is a previously unrecognized novel function of these permeases. In addition to molybdate, other oxyanions, like sulfate, tungstate, and vanadate, are likely to be imported by the R. capsulatus permease.     

A sulfate,sulfite and thiosulfate incorporating system inCandida utilis

Sulfate, sulfite and thiosulfate incorporation in the yeastCandida utilis is inhibited by extracellular sulfate, sulfite and thiosulfate and by sulfate analogues selenate, chromate and molybdate. The three processes are blocked if sulfate, sulfite, thiosulfate, cysteine and homocysteine are allowed to accumulate endogenously. Incorporation of the three inorganic sulfur oxy anions is inactivated by heat at the same rate. Mutants previously shown to be defective in sulfate incorporation are also affected in sulfite and thiosulfate uptake. Revertants of these mutants selected by plating in ethionine-supplemented minimal medium recovered the capacity to incorporate sulfate, sulfite and thiosulfate. These results taken together with previous evidence demonstrate the existence of a common sulfate, sulfite and thiosulfate incorporating system in this yeast.     

Inhibition of phosphatase and sulfatase by transition-state analogues

The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase. Vanadate was a potent inhibitor of all three enzymes. Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-). The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase. Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions. Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase. Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol. It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes. The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed.     

Removal of selenate from sulfate-containing media by sulfate-reducing bacterial biofilms

A biofilm-selected strain of a Desulfomicrobium sp. removed selenate from solution to sub-micromolar concentrations during growth on lactate (or hydrogen) and sulfate. Under sulfate-limited growth conditions, selenium was enzymatically reduced to selenide. Under excess sulfate conditions, selenate removal was primarily by enzymatic reduction to elemental selenium. Sequestration by biofilms was greater under the latter condition. Experiments with washed cell suspensions showed that high sulfate concentrations inhibited cell-specific selenate reduction, but when growing cells were exposed to selenate, the biomass increase achieved during incubations with abundant sulfate resulted in more rapid selenate removal. The addition of small amounts of sulfite, or thiosulfate, ameliorated this inhibition. Nitrate also inhibited selenate reduction in washed cell suspensions, apparently due to a general oxidizing effect. These results suggest that where biofilm-based sulfate-reducing bacteria (SRB) bioreactors are considered for the treatment of mixed metalliferous wastes that contain selenium oxyanions, adequate selenate removal should be achievable under a range of environmental conditions. The form and fate of the precipitated product will, however, be influenced by the dominant reduction pathway, which is controlled by environmental variables.     

Chromate and selenate hydrocalumite solid solutions and their applications in waste treatment

Hydrocalumite, a calcium aluminate hydrate phase, consists of positively-charged structure units, and is therefore an ideal candidate for accommodating anionic contaminants. In this study, a series of batch experiments was carried out to examine the uptake of chromate and selenate by hydrocalumite. To determine the uptake capacity and long-term stability, hydro-calumite solid solutions between chromate/selenate and hydroxyl were synthesized over a reaction time of more than one year. At a ratio of water to initial solids added (CaAl2O4+CaO) of 75 : 1, the maximum uptake capacities were over 77 and 114 g/kg for Cr and Se, respectively. These values are very close to the theoretical uptake capacities of chromate and selenate hydrocalumite end-members (81 and 118 g/kg, respectively). The oxyanion removal efficiency from solution was above 95%. Due to the high uptake capacity and anion removal efficiency of hy-drocalumites, their application in wastewater treatment is promising. Hydrocalumites are also impor     

Chromate and selenate hydrocalumite solid solutions and their applications in waste treatment

Hydrocalumite, a calcium aluminate hydrate phase, consists of positively-charged structure units, and is therefore an ideal candidate for accommodating anionic contaminants. In this study, a series of batch experiments was carried out to examine the uptake of chromate and selenate by hydrocalumite. To determine the uptake capacity and long-term stability, hydrocalumite solid solutions between chromate/selenate and hydroxyl were synthesized over a reaction time of more than one year. At a ratio of water to initial solids added (CaAl2O4+CaO) of 75: 1, the maximum uptake capacities were over 77 and 114 g/kg for Cr and Se, respectively.These values are very close to the theoretical uptake capacities of chromate and selenate hydrocalumite end-members (81 and 118 g/kg, respectively). The oxyanion removal efficiency from solution was above 95%. Due to the high uptake capacity and anion removal efficiency of hydrocalumites, their application in wastewater treatment is promising. Hydrocalumites are also important hydration products of cementitious materials and the long-term stability of these phases is of significance for application in solidification/stabilization technology.     

Identification of intrinsic high-level resistance to rare-earth oxides and oxyanions in members of the class Proteobacteria: characterization of tellurite, selenite, and rhodium sesquioxide reduction in Rhodobacter sphaeroides.

We have identified intrinsic high-level resistance (HLR) to tellurite, selenite, and at least 15 other rare-earth oxides and oxyanions in the facultative photoheterotroph Rhodobacter sphaeroides grown either chemoheterotrophically or photoheterotrophically. Other members of the class Proteobacteria, including members of the alpha-2 and alpha-3 phylogenetic subgroups, were also shown to effect the reduction of many of these compounds, although genera from the alpha-1, beta-1, and gamma-3 subgroups did not express HLR to the oxyanions examined. Detailed analyses employing R. sphaeroides have shown that HLR to at least one class of these oxyanions, the tellurite class (e.g., tellurate, tellurite, selenate, selenite, and rhodium sesquioxide), occurred via intracellular oxyanion reduction and resulted in deposition of metal in the cytoplasmic membrane. The concomitant evolution of hydrogen gas from cells grown photoheterotrophically in the presence of these oxyanions was also observed. HLR to tellurite class oxyanions in R. sphaeroides was not affected by exogenous methionine or phosphate but was reduced 40-fold by the addition of cysteine to growth media. In contrast HLR to the periodate class oxyanions (e.g., periodate, siliconate, and siliconite) was inhibited by extracellular PO4(3-) but did not result in metal deposition or gas evolution. Finally, we observed that HLR to arsenate class oxyanions (e.g., arsenate, molybdate, and tungstate) occurred by a third, distinct mechanism, as evidenced by the lack of intracellular metal deposition and hydrogen gas evolution and an insensitivity to extracellular PO4(3-) or cysteine. Examination of a number of R. sphaeroides mutants has determined the obligate requirement for an intact CO2 fixation pathway and the presence of a functional photosynthetic electron transport chain to effect HLR to K2TeO3 under photosynthetic growth conditions, whereas functional cytochromes bc1 and c2 were required under aerobic growth conditions to facilitate HLR. Finally, a purification scheme to recover metals from intact bacterial cells was developed.     

Synechococcus elongatus PCC 7942 is more tolerant to chromate as compared to Synechocystis sp. PCC 6803

Two unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 showed contrasting responses to chromate stress with EC₅₀ of 12 ± 2 and 150 ± 15 μM potassium dichromate respectively. There was no depletion of chromate in growth medium in both the cases. Using labeled chromate, very low accumulation (<1 nmol/10⁸ cells) was observed in Synechocystis after incubation for 24 h in light. No accumulation of chromate could be observed in Synechococcus under these conditions. Chromate oxyanion is known to enter the cells using sulfate uptake channels. Therefore, inhibition of sulfate uptake caused by chromate was monitored using ³⁵S labeled sulfate. IC₅₀ values of chromate for ³⁵sulfate uptake were higher in Synechococcus as compared to Synechocystis. The results suggested that the sulfate transporters in Synechococcus have lower affinity to chromate than those from Synechocystis possibly due to differences in affinity of sulfate receptors for chromate. Bioinformatic analyses revealed presence of sulfate and chromate transporters with considerable similarity; however, minor differences in these may play a role in their differential response to chromate. In both cases the IC₅₀ values decreased when sulfate concentration was reduced in the medium indicating competitive inhibition of sulfate uptake by chromate. Interestingly, Synechococcus showed stimulation of growth at concentrations of chromate less than 100 μM, which affected its cell size without disturbing the ultrastructure and thylakoid organization. In Synechocystis, growth with 12 μM potassium dichromate damaged the ultrastructure and thylakoid organization with slight elongation of the cells. The results suggested that Synechococcus possesses efficient strategies to prevent entry and to remove chromate from the cell as compared to Synechocystis. This is the first time a differential response of Synechococcus 7942 and Synechocystis 6803 to chromate is reported. The contrasting characteristics observed in the two cyanobacteria will be useful in understanding the basis of resistance or susceptibility to chromate.     

Chromate and selenate hydrocalumite solid solutions and their applications in waste treatment

Hydrocalumite, a calcium aluminate hydrate phase, consists of positively-charged structure units, and is therefore an ideal candidate for accommodating anionic contaminants. In this study, a series of batch experiments was carried out to examine the uptake of chromate and selenate by hydrocalumite. To determine the uptake capacity and long-term stability, hydrocalumite solid solutions between chromate/selenate and hydroxyl were synthesized over a reaction time of more than one year. At a ratio of water to initial solids added (CaAI₂O₄+CaO) of 75:1, the maximum uptake capacities were over 77 and 114 g/kg for Cr and Se, respectively. These values are very close to the theoretical uptake capacities of chromate and selenate hydrocalumite end members (81 and 118 g/kg, respectively). The oxyanion removal efficiency from solution was above 95%. Due to the high uptake capacity and anion removal efficiency of hydrocalumites, their application in wastewater treatment is promising. Hydrocalumites are also important hydration products of cementitious materials, the long-term stability of these phases is of significance for application in solidification/stabilization technology.