High Concentrations Of Molybdate Research Articles

New Family of Tungstate-Responsive Transcriptional Regulators in Sulfate-Reducing Bacteria

The trace elements molybdenum and tungsten are essential components of cofactors of many metalloenzymes. However, in sulfate-reducing bacteria, high concentrations of molybdate and tungstate oxyanions inhibit growth, thus requiring the tight regulation of their homeostasis. By a combination of bioinformatic and experimental techniques, we identified a novel regulator family, tungstate-responsive regulator (TunR), controlling the homeostasis of tungstate and molybdate in sulfate-reducing deltaproteobacteria. The effector-sensing domains of these regulators are similar to those of the known molybdate-responsive regulator ModE, while their DNA-binding domains are homologous to XerC/XerD site-specific recombinases. Using a comparative genomics approach, we identified DNA motifs and reconstructed regulons for 40 TunR family members. Positional analysis of TunR sites and putative promoters allowed us to classify most TunR proteins into two groups: (i) activators of modABC genes encoding a high-affinity molybdenum and tungsten transporting system and (ii) repressors of genes for toluene sulfonate uptake (TSUP) family transporters. The activation of modA and modBC genes by TunR in Desulfovibrio vulgaris Hildenborough was confirmed in vivo, and we discovered that the activation was diminished in the presence of tungstate. A predicted 30-bp TunR-binding motif was confirmed by in vitro binding assays. A novel TunR family of bacterial transcriptional factors controls tungstate and molybdate homeostasis in sulfate-reducing deltaproteobacteria. We proposed that TunR proteins participate in protection of the cells from the inhibition by these oxyanions. To our knowledge, this is a unique case of a family of bacterial transcriptional factors evolved from site-specific recombinases.

Characterisation of the anodic layers formed on 2024 aluminium alloy, in tetraborate electrolyte containing molybdate ions

Anodic layer growth on 2024 aluminium alloy at 70 °C, under 40 V, during 60 min, in 50 g L −1 di-sodium tetraborate solution containing di-sodium molybdate from 0.1 to 0.5 M (pH 10) is examined. Anodising behaviours strongly depend on additive concentration. Development of anodic films is favoured with weak molybdate additions (<0.3–0.4 M). The film thicknesses increase and the porosity of anodic layers decreases. Molybdenum (+VI), detected by X-ray photoelectron spectroscopy (XPS) analysis, is present in the anodic films and the Mo incorporation, studied by energy dispersive spectroscopy (EDS) analysis, increases with molybdate concentration. However, for high molybdate concentrations (>0.4 M), anodising behaviour becomes complex with the formation of a blue molybdenum oxide at the cathode. The growth of aluminium oxide is hindered. As the anodic layers are thinner, the Mo(+VI) incorporation significantly decreases. These two configurations implicate different corrosion performances in 5% sodium chloride solution at 35 °C. As the alkaline anodic layer formed with 0.3 M molybdate species is the thickest and the Mo incorporation is the more pronounced, its corrosion resistance is the highest. The effect of morphology and composition of anodic films on pitting corrosion is also discussed.

In Vitro Molybdenum Ligation to Molybdopterin Using Purified Components

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.

Electrodeposited cobaltmolybdenum magnetic materials

Cobaltmolybdenum (CoMo) induced electrodeposition has been studied from a sulphate+citrate bath on carbon electrodes, under conditions near to neutral pH. Crack-free homogeneous deposits with a low percentage of molybdenum can be easily obtained from low molybdate concentrations applying low deposition potentials or current densities. The formation of this kind of deposit is related to Q ox/ Q red around 1 in the voltammetric/stripping experiments. Moreover, percentages of molybdenum up to 60% can be obtained from high molybdate concentrations but, in this case, the deposits show cracks. The formation of these cracked deposits can be predicted from the observed distortions in the j– t and E– t deposition transients. Coatings with a partially amorphous structure, or with a crystalline structure of nanometric crystal size, are obtained. The magnetisation results show that the saturation magnetisation gradually decreases when the percentage of molybdenum increases in the deposit. Simultaneously, a clear decrease of the coercitivity is observed from the lowest percentage of molybdenum with respect to the value of pure cobalt coatings.

In Vitro Incorporation of Nascent Molybdenum Cofactor into Human Sulfite Oxidase

We were able to reconstitute molybdopterin (MPT)-free sulfite oxidase in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate. MPT-free human sulfite oxidase apoprotein was obtained by heterologous expression in an Escherichia coli mutant with a defect in the early steps of MPT biosynthesis. In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor Z, purified MPT synthase, and sodium molybdate. In vitro synthesized MPT generated from precursor Z by MPT synthase remains bound to the synthase. Surprisingly, MPT synthase was found capable of donating bound MPT to MPT-free sulfite oxidase. MPT was not released from MPT synthase when either bovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase. After the inclusion of sodium molybdate in the reconstitution mixture, active sulfite oxidase was obtained, revealing that in vitro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presence of high concentrations of molybdate. The conversion of MPT into Moco by molybdate chelation apparently occurs concomitantly with the insertion of MPT into sulfite oxidase.

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

ABSTRACTStrain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM. However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.

Molybdenum cofactor biosynthesis in Escherichia coli mod and mog mutants.

The molybdopterin content of Escherichia coli mod and mog mutants was estimated by conversion to the form A derivative. The results are in accord with complete phenotypic repair of mod, and incomplete repair of mog, by culture in high concentrations of molybdate. A possible role for Mog as a molybdochelatase is discussed.

Molybdenum co‐factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two‐domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1, a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA. The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum co-factor locus chl6. As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli, is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.

Nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.

Isolation of Escherichia coli mutants defective in uptake of molybdate.

For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.

Electron donors and the quinone involved in dimethyl sulfoxide reduction inEscherichia coli

Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehydrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of thechlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants ofE. coli blocked in menaquinone (vitamin K2) biosynthesis—menB, menC, andmenD—were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates,o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.

Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli.

Escherichia coli used tetrahydrothiophene 1-oxide (THTO) as an electron acceptor for anaerobic growth with glycerol as a carbon source; the THTO was reduced to tetrahydrothiophene. Cell extracts also reduced THTO to tetrahydrothiophene in the presence of a variety of electron donors. Chlorate-resistant (chl) mutants (chlA, chlB, chlD, and chlE) were unable to grow with THTO as the electron acceptor. However, growth and THTO reduction by the chlD mutant were restored by high concentrations of molybdate. Similarly, mutants of E. coli that are blocked in the menaquinone (vitamin K2) biosynthetic pathway, i.e., menB, menC, and menD mutants, did not grow with THTO as an electron acceptor. Growth and THTO reduction were restored in these mutants by the presence of appropriate intermediates of the vitamin K biosynthetic pathway.

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.

Resolution of non-activated and activated androgen receptors based on differences in their hydrodynamic properties

This study shows that cytosolic androgen receptor of rat ventral prostate sediments at 10–11 S on conventional low salt sucrose density gradients (SDG), and at 4.6 S on high salt SDG, whether it is activated or not; inclusion of 10mM Na 2MoO 4 in all buffers does not alter these sedimentation coefficients. In the presence of 50 mM Na 2MoO 4 non-activated and activated androgen receptors sediment in high salt SDG at 7–8 S and 4.6 S, respectively. Thus the presence of high concentrations of molybdate during centrifugation inhibits the KCl induced disaggregation of receptor into subunits. Similar effects are observed on Sephacryl-S200 gel filtration; in 50 mM MoO 4 2− and 0.4 M KCl non-activated receptor has an estimated Stokes radius of 67 Å; this value decreases to 52 Å upon activation in the presence of proteolysis inhibitors; omission of molybdate during chromatography yielded 52 Å and 27 Å entities. Estimated mol. wts are 198,000 Daltons for the non-activated 67 Å form and 98,000 Daltons for the activated 52 Å receptor. Sodium molybdate (50 mM) prevents temperature (18°C) and high ionic strength (0.4 M KCl) induced receptor activation. This inhibition was overcome by removing molybdate by centrifugal gel filtration, or by increasing the KCl concentration to 0.8 M. The inhibitory effects of molybdate on salt induced receptor disaggregation into activated subunits are no longer observed at pH 7.4 or after chemical modification of sulfhydryl groups. Once androgen receptor has been disaggregated into its activated subunits the activated state is maintained even upon reassociation to 10–11 S aggregates in low salt. The relative concentrations of KC1 and molybdate are critical; thus, 10 mM Na 2MoO 4/0.4M KCl and 50 mM Na 2MoO 4/0.8–1.2M KCl did not differentiate activated from non-activated androgen receptor based on their hydrodynamic properties. In the presence of 0.4 M KCl and 50 mM molybdate, however, the hydrodynamic properties of androgen receptor can be correlated with receptor activation.

Initial and residual effectiveness of molybdate fertilizer in two areas of south western Australia

A range of levels of sodium molybdate was applied to existing clover pastures at Boyup Brook and at Bakers Hill in south-western Australia. In the three subsequent years, further levels of molybdate were applied to some of the sites to assess the residual value of the initial application. The effect of the molybdate application on the molybdenum concentration in clover tops was measured. Plots of molybdenum concentration in tops versus molybdate applied curved upwards at first as each additional increment provided an increased response in concentration. However, at high concentrations of molybdate in clover tops the plots curved to the right at three out of four sites as each additional increment produced a decreased response. That is, overall, the curves were sigmoid. Increases in molybdate concentration were greatest on soils with least ability to retain molybdate as measured in the laboratory. Molybdate applied one year previously was about half as effective as currently applied molybdate. After two and three years it was about 20% as effective. A single application of rnolybdate remains effective for a long period on a soil of low ability to retain molybdate because the initial application is super-abundant - rather than because of large differences between soils in the rate of decline of effectiveness.

Biochemical and genetical characterization of nitrate reductase deficient mutants of Petunia

Four NR(-) lines were selected by their resistance to 100 mM chlorate from X-ray irradiated protoplasts of haploid Petunia hybrida var. Mitchell. The four cell lines were characterized by the presence of xanthine dehydrogenase activity and by complementation tests via protoplast fusion. One mutant (line 1) was classified as defective in the NR apoprotein (tentatively, nia-type) and the other three (lines 2, 3, 4) in the molybdenum cofactor (tentatively, cnx-type). Some NR activity (15 %) could be restored by adding unphysiologically high concentrations of molybdate to the culture medium in two of the cnx-lines (lines 3 and 4). The third cnx-line (line 2) had no NR activity. A complementation analysis via protoplast fusion confirmed that the mutants comprised 3 non-allelic groups. From these results it can be concluded that these NR(-) mutants are recessive and that two of the cnx-mutants (lines 3, 4) are allelic.

Measurement of phosphate concentration in the presence of very labile phosphate esters

There are a number of methods available for the measurement of phosphate ion concentration, which may be used when moderately labile phosphate esters such as ATP are present in low concentration. However, the highly acidic conditions usually employed make these unsuitable when very labile esters such as phosphocreatine are present. A method in which the phosphomolybdate complex is developed under mildly acidic conditions, using high molybdate concentrations to counteract the reduced assay sensitivity at high pH, is described. The assay is linear in the range 5–300 μ m phosphate, and micromolar concentrations of phosphate can be reliably measured in the presence of millimolar phosphocreatine.

Transformation of human androgen receptors to the deoxyribonucleic acid-binding state.

The transformation of [3H]dihydrotestosterone-androgen receptor complexes to the nuclear binding state was studied in fibroblasts cultured from human genital skin, using binding of the androgen-receptor complex to DNA-cellulose as a measure of transformation. Transformation of cytosolic dihydrotestosterone-receptor complexes proceeded slowly at 0 C, and heating to 25 C caused rapid transformation that was essentially complete within 15 min. Sodium molybdate (up to 10 mM) slowed the rate of transformation in a concentration-dependent fashion; low concentrations of molybdate (0.5-1 mM) prevented transformation at 0 C, but transformation of up to 70% of the total cytosolic hormone-receptor complexes could be achieved in the presence of high concentrations of molybdate by appropriately prolonged heating at 25 C. Molybdate appeared to exhibit a dual effect, increasing the total amount of androgen-receptor complex recoverable in cytosol and prolonging the time course of heat-induced transformation. Treatment of cytosolic dihydrotestosterone-receptor complexes with 0.5 M NaCl caused a degree of transformation similar to that seen after heat treatment but was unaffected by 10 mM molybdate. Transformation was accompanied by disappearance of the 7S form of the receptor on sucrose density gradients, but there was no reciprocal increase in a smaller size binding moiety on low ionic strength sucrose density gradients. The heat-transformed receptor could be detected, however, as a 3S binding peak on gradients containing 0.3 M NaCl, suggesting that transformation was accompanied by a structural alteration of the hormone-receptor complex.

Regeneration of fully nitrate reductase — deficient mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Protoplast-derived colonies of haploid N. plumbaginifolia were selected for by chlorate resistance in media supplemented with casamino acids. Eighty resistant lines were confirmed by a second passage on a higher concentration of chlorate. Frequency of spontaneous mutation ranged from 10(-5) to 10(-6). Fifty of the resistant lines could be regenerated into plants, and 30 were characterized biochemically. Ninety percent were fully deficient for nitrate reductase activity. The lines were further tested for xanthine dehydrogenase activity and subsequently classified as defective in the apoenzyme (nia type, 26 lines) or the cofactor (cnx type, 4 lines). Two groups had been identified up until now within the cnx type by growth tests on high concentrations of molybdate supplied to the medium. Nitrate reductase deficiency was stably and continously expressed in both variant cell cultures and regenerants. Genetic analysis demonstrated that nitrate reductase deficiency was inherited as a single recessive nuclear gene.

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.