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.

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.

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.

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.