Sulfite oxidation catalyzed by aa(3)-type cytochrome c oxidase in Acidithiobacillus ferrooxidans.

Sulfite is produced as a toxic intermediate during Acidithiobacillus ferrooxidans sulfur oxidation. A. ferrooxidans D3-2, which posseses the highest copper bioleaching activity, is more resistant to sulfite than other A. ferrooxidans strains, including ATCC 23270. When sulfite oxidase was purified homogeneously from strain D3-2, the oxidized and reduced forms of the purified sulfite oxidase absorption spectra corresponded to those of A. ferrooxidans aa(3)-type cytochrome c oxidase. The confirmed molecular weights of the α-subunit (52.5 kDa), the ß-subunit (25 kDa), and the γ-subunit (20 kDa) of the purified sulfite oxidase and the N-terminal amino acid sequences of the γ-subunit of sulfite oxidase (AAKKG) corresponded to those of A. ferrooxidans ATCC 23270 cytochrome c oxidase. The sulfite oxidase activities of the iron- and sulfur-grown A. ferrooxidans D3-2 were much higher than those cytochrome c oxidases purified from A. ferrooxidans strains ATCC 23270, MON-1 and AP19-3. The activities of sulfite oxidase purified from iron- and sulfur-grown strain D3-2 were completely inhibited by an antibody raised against a purified A. ferrooxidans MON-1 aa(3)-type cytochrome c oxidase. This is the first report to indicate that aa(3)-type cytochrome c oxidase catalyzed sulfite oxidation in A. ferrooxidans.

Volatilization of metal mercury from Organomercurials by highly mercury-resistant Acidithiobacillus ferrooxidans MON-1.

The iron-oxidizing bacterium Acidithiobacillus ferrooxidans MON-1 is highly resistant not only to mercuric chloride (HgCl(2)) but also to organomercurials such as methylmercury chloride (MMC). We have found that cytochrome c oxidase, purified from strain MON-1, reduces Hg(2+) to volatilizable metal mercury (Hg(0)) with reduced mammalian cytochrome c or Fe(2+) as an electron donor. In this study we found that cytochrome c oxidase can volatilize Hg(0) from MMC as well as from Hg(2+) with reduced mammalian cytochrome c or c-type cytochrome purified from strain MON-1 as an electron donor. We also found that MMC-Hg(0) volatilization activity is present in the MON-1 plasma membrane but not in the cytosol. These activities were strongly inhibited by sodium cyanide (NaCN) and the antibody produced against purified MON-1 cytochrome c oxidase. This is the first report to indicate that cytochrome c oxidase is involved in the degradation of organomercurials in microorganisms.

Ferrous iron production mediated by tetrathionate hydrolase in tetrathionate-, sulfur-, and iron-grown Acidithiobacillus ferrooxidans ATCC 23270 cells.

When tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells were incubated with ferric ions and tetrathionate at pH 3.0, ferrous ions were produced enzymatically. Fe(3+)-reductase, which catalyzes Fe(3+) reduction with tetrathionate, was purified to homogeneity not only from tetrathionate-grown, but also from sulfur- and iron-grown A. ferrooxidans ATCC 23270 cells. The results for apparent molecular weight measured by SDS-PAGE (52.3 kD) and the N-terminal amino acid sequences of the purified enzymes from iron-, sulfur, and tetrathionate-grown cells (AVAVPMDSTG) indicate that Fe(3+)-reductase corresponds to tetrathionate hydrolase. The evidence that tetrathionate-grown A. ferrooxidans ATCC 23270 cells have high iron-oxidizing activity at the early log phase, comparable to that of iron-grown ATCC 23270 cells, is supported by our finding that tetrathionate hydrolase produces Fe(2+) from tetrathionate during growth on tetrathionate. This is the first report on ferric reductase activity associated with tetrathionate hydrolase.

Reconstitution of iron oxidase from sulfur-grown Acidithiobacillus ferrooxidans.

The iron oxidation system from sulfur-grown Acidithiobacillus ferrooxidans ATCC 23270 cells was reconstituted in vitro. Purified rusticyanin, cytochrome c, and aa(3)-type cytochrome oxidase were essential for reconstitution. The iron-oxidizing activity of the reconstituted system was 3.3-fold higher than that of the cell extract from which these components were purified.

Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1.

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.

Isolation and characterization of Acidithiobacillus ferrooxidans strain D3-2 active in copper bioleaching from a copper mine in Chile.

Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu(2+) solubilized from 1% chalcopyrite (CuFeS(2)) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg x l(-1) x 30 d(-1). The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 microl O(2) uptake x mg protein(-1) x min(-1). In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 mul O(2) uptake.mg protein(-1).min(-1). Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.

Increase in Fe2+-producing activity during growth of Acidithiobacillus ferrooxidans ATCC23270 on sulfur.

When Acidithiobacillus ferrooxidans ATCC23270 cells, grown for many generations on sulfur were grown in sulfur medium with and without Fe(3+), the bacterium markedly increased not only in iron oxidase activity but also in Fe(2+)-producing sulfide:ferric ion oxidoreductase (SFORase) activity during the early log phase, and retained part of these activities during the late log phase. The activity of SFORase, which catalyzes the production of Fe(2+) from Fe(3+) and sulfur, of sulfur-grown cells was approximately 10-20 fold higher than that of iron-grown cells. aa(3) type cytochrome c oxidase, an important component of iron oxidase in A. ferrooxidans, was partially purified from sulfur-grown cells. A. ferrooxidans ATCC23270 cells grown for many generations on sulfur had the ability to grow on iron as rapidly as that did iron-grown cells. These results suggest that both iron oxidase and Fe(2+)-producing SFORase have a role in the energy generation of A. ferrooxidans ATCC23270 from sulfur.

Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans.

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

Volatilization and recovery of mercury from mercury-polluted soils and wastewaters using mercury-resistant Acidithiobacillus ferrooxidans strains SUG 2-2 and MON-1.

Iron-oxidizing bacterium, Acidithiobacillus ferrooxidans, is one of the most important bacteria for the bioleaching of copper and gold ores. In order to use the mercury reducing activity of A. ferrooxidans for the bioremediation of mercury, mercury-resistant A. ferrooxidans strains SUG 2-2 and MON-1 were screened among 150 strains of iron-oxidizing bacteria isolated from natural environments. It was found that strains SUG 2-2 and MON-1 have a novel ferrous iron-dependent mercury volatilization activity as well as an NADPH-dependent mercury reductase activity. Strain MON-1 has an organomercurial lyase-like activity and grew most rapidly in an iron medium with 0.1 microM p-chloromercuribenzoic acid among 11 A. ferrooxidans strains tested. Nearly 100% of the total mercury in mercury-polluted soil or mercury wastewater was volatilized and recovered by incubating SUG 2-2 or MON-1 cells in 20 ml of an acidified water (pH 2.5) with ferrous iron, suggesting that these mercury-resistant strains can be used for the bioremediation of inorganic and organic mercurial compounds. We show for the first time that MON-1 cells immobilized in polyvinyl alcohol (PVA) resins could efficiently volatilize mercury from 2 L of a synthetic mercury-polluted wastewater (pH 2.5) containing 40 microM Hg(2+) and ferrous iron. The MON-1-immobilized PVA resins were used repeatedly.

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans.

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.

Existence of an iron-oxidizing bacterium Acidithiobacillus ferrooxidans resistant to organomercurial compounds.

Acidithiobacillus ferrooxidans MON-1 which is highly resistant to Hg2+ could grow in a ferrous sulfate medium (pH 2.5) with 0.1 microM p-chloromercuribenzoic acid (PCMB) with a lag time of 2 d. In contrast, A. ferrooxidans AP19-3 which is sensitive to Hg2+ did not grow in the medium. Nine strains of A. ferrooxidans, including seven strains of the American Type Culture Collection grew in the medium with a lag time ranging from 5 to 12 d. The resting cells of MON-1, which has NADPH-dependent mercuric reductase activity, could volatilize Hg0 when incubated in acidic water (pH 3.0) containing 0.1 microM PCMB. However, the resting cells of AP19-3, which has a similar level of NADPH-dependent mercuric reductase activity compared with MON-1, did not volatilize Hg0 from the reaction mixture with 0.1 microM PCMB. The activity level of the 11 strains of A. ferrooxidans to volatilize Hg0 from PCMB corresponded well with the level of growth inhibition by PCMB observed in the growth experiments. The resting cells of MON-1 volatilized Hg0 from phenylmercury acetate (PMA) and methylmercury chloride (MMC) as well as PCMB. The cytosol prepared from MON-1 could volatilize Hg0 from PCMB (0.015 nmol mg(-1) h(-1)), PMA (0.33 nmol mg(-1) h(-1)) and MMC (0.005 nmol mg(-1) h(-1)) in the presence of NADPH and beta-mercaptoethanol.

Existence of a tungsten-binding protein in Acidithiobacillus ferrooxidans AP19-3.

A tungsten-binding protein was purified from a plasma membrane preparation of the iron-oxidizing bacterium, Acidithiobacillus ferrooxidans AP19-3 in an electrophoretically homogenous state. The protein was composed of two subunits with apparent molecular masses of 12 and 20.7 kDa. The molecular mass of the native protein was estimated to be 26.4 kDa in the presence of 1.5% 1-o-octyl-D -glucopyranoside (OGL), indicating that the native tungsten-binding protein is a heterodimeric protein. The amounts of tungsten bound to 1 mg of plasma membranes of A. ferrooxidans AP19-3 and the purified tungsten-binding protein at pH 3.0 were 191 and 1506 mug, respectively. In contrast, the amounts of tungsten bound to 1 mg of albumin, aldolase, catalase, chymotrypsinogen A, ferritin, and ferredoxin at pH 3.0 were 13.1, 18.6, 12.8, 16.6, 11.4, and 6.1 mug, respectively. Incubation of the tungsten-binding protein for 1 h with 10 mM Na(2)WO(4) plus 10 mM metal ion, such as NaVO(3), Na(2)MoO(4), CuSO(4), NiSO(4), MnSO(4), CoSO(4), or CdCl(2), did not markedly affect the amount of tungsten bound to the tungsten-binding protein, suggesting that the protein specifically binds tungsten.

Noncompetitive inhibition by L-cysteine and activation by L-glutamate of the iron-oxidizing activity of a mixotrophic iron-oxidizing bacterium strain OKM-9.

A mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9 uses ferrous iron as a sole source of energy and L-glutamate as a sole source of cellular carbon. Uptake of L-glutamate into OKM-9 cells is absolutely dependent on ferrous iron oxidation. Thus, the Fe(2+)-dependent L-glutamate uptake system of strain OKM-9 is crucial for the bacterium to grow mixotrophically in iron medium with L-glutamate. The relationship between iron oxidation and L-glutamate transport activities was studied. Iron oxidase containing cytochrome a was purified 9-fold from the plasma membrane of OKM-9. A purified iron oxidase showed one rust-colored band following disc gel electrophoresis after incubation with Fe(2+). The Fe(2+)-dependent L-glutamate transport system was also purified 14.5-fold from the plasma membrane using the same purification steps as for iron oxidase. Fe(2+)-dependent L-glutamate and L-cysteine uptake activities of OKM-9 were 0.36 and 0.24 nmol/mg/min, respectively, when a concentration of 18 mM of these amino acids was used as a substrate. Both uptake activities were completely inhibited by potassium cyanide (KCN), suggesting that cytochrome a in the iron oxidase is involved in the transport process. The iron-oxidizing activity of strain OKM-9 was activated 1.7-fold by 80 mM L-glutamate. In contrast, the activity was noncompetitively inhibited by L-cysteine. The Michaelis constant of iron oxidase for Fe(2+) was 12.6 mM and the inhibition constant for L-cysteine was 41.6 mM. A marked inhibition of iron oxidase by 50 mM L-cysteine was completely reversed by the addition of 60 mM L-glutamate. The results suggest the possibility that iron oxidase has a binding site for L-cysteine and the cysteine first bound to the iron oxidase was replaced by the added L-glutamate.

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1.

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.

Volatilization and recovery of mercury from mercury wastewater produced in the course of laboratory work using Acidithiobacillus ferrooxidans SUG 2-2 cells.

The iron-oxidizing bacterium Acidithiobacillus ferrooxidans SUG 2-2 is markedly resistant to mercuric chloride and can volatilize mercury (Hg0) from mercuric ion (Hg2+) under acidic conditions. To develop a microbial technique to volatilize and recover mercury from acidic and organic compound-containing mercury wastewater, which is usually produced in the course of everyday laboratory work in Okayama University, the effects of organic and inorganic chemicals on the mercury volatilization activity of A. ferrooxidans cells were studied. Among 55 chemicals tested, the mercury volatilization from a reaction mixture (pH 2.5) containing resting cells of SUG 2-2 (1 mg of protein) and mercury chloride (14 nmol) was strongly inhibited by AgNO3 (0.05 mM), K2CrO7 (1.0 mM), cysteine (1.0 mM), trichloroethylene (1 microM), and commercially produced detergents (0.05%). However, the strong inhibition by trichloroethylene and detergents was not observed when these organic compounds were chemically decomposed using Fenton's method before the treatment of the wastewater with SUG 2-2 cells. When 20 ml of water acidified with sulfuric acid (pH 2.5) containing ferrous sulfate (3%), diluted mercury wastewater (17.5 nmol of Hg2+) and SUG 2-2 cells (0.05 mg of protein) were incubated for 10 d at 30 degrees C, 47% of the total mercury in the wastewater was volatilized and recovered into a trapping reagent for metal mercury. However, when the organic compounds in the mercury wastewater were decomposed using Fenton's method and then treated with A. ferrooxidans cells, approximately 100% of the total mercury in the wastewater was volatilized and recovered.