Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: Purification and properties

Abstract Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans . It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, E _m,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c , cytochrome c -550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c -552 (m) and c -55 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c -550 (m) was higher than that of cytochrome c -552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c -552 (m) was greatly lower than that of cytochrome c -552 (s).     

Continuous bacterial ferrous iron oxidation by Thiobacillus ferrooxidans in rotating biological contactors

Summary Fe oxidation in rotating biological contactors has been studied over a range of influent Fe concentrations. Rotation speeds greater than 20 rpm did not affect the oxidation rate. Hydraulic loading rates above a critical value reduce the oxidation rat at influent Fe>4g/L.     

Adaptation of Thiobacillus ferrooxidans to nickel ion and bacterial oxidation of nickel sulfide

Summary The Ni²⁺ resistance of Thiobacillus ferrooxidans was enhanced by repeated culturing in medium containing Ni²⁺ and gradually increasing the Ni²⁺ concentration. The extraction of nickel sulfide was enhanced by the adapted strain following the direct leaching mechanism of the microorganism.     

Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much more intensely for epoxide hydrolase and glutathione S-transferases B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to epoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Sequence variability in bacterial cytochromes c.

Cytochromes c are proteins that can be defined both phenotypically and by their possession of a characteristic sequence motif. Many sequences from bacterial sources are known, and new ones are being reported every year. An analysis can be made as to what fraction of new sequences are members of already known classes or subclasses, and how many map into previously uninhabited regions of sequence space.     

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.     

Stannous and cuprous ion oxidation by Thiobacillus ferrooxidans.

Oxidation of stannous chloride by Thiobacillus ferrooxidans was studied manometrically. At low stannous ion concentrations, initial oxidation rate was proportional to concentration. Optimum pH for oxidation was 2.3 optimum temperature was 37-40 degrees C. Spectrophotometry showed reduction of cytochromes in suspensions of whole cells on addition of ferrous, stannous, or cuprous salts. Cytochrome c reductase activity in cell-free extracts was assayed with ferrous, stannous, or cuprous ions as electron donors. It appears unlikely that an essential non-biological reaction, the reduction of ferric ions by stannous or cuprous ions, is involved. Growth of T. ferrooxidans was not obtained with either stannous chloride or stannous sulphate as sole energy source.     

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.     

Identification of c-type cytochromes involved in anaerobic, bacterial U(IV) oxidation

Anaerobic, bacterial reduction of water-soluble U(VI) complexes to the poorly soluble U(IV) mineral uraninite has been intensively studied as a strategy for in situ remediation of uranium-contaminated groundwater. A novel and potentially counteracting metabolic process, anaerobic, nitrate-dependent U(IV) oxidation, has recently been described in two bacterial species (Geobacter metallireducens and Thiobacillus denitrificans), but the underlying biochemistry and genetics are completely unknown. We report here that two diheme, c-type cytochromes (putatively c ₄ and c ₅ cytochromes) play a major role in nitrate-dependent U(IV) oxidation by T. denitrificans. Insertion mutations in each of the two genes encoding these cytochromes resulted in a greater than 50% decrease in U(IV) oxidation activity, and complementation in trans restored activity to wild-type levels. Sucrose-density-gradient ultracentrifugation confirmed that both cytochromes are membrane-associated. Insertion mutations in genes encoding other membrane-associated, c-type cytochromes did not diminish U(IV) oxidation. This is the first report of proteins involved in anaerobic U(IV) oxidation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction kinetics of bacterial cytochromes c2.

In a continuing effort to understand the mechanism of electron transfer by c-type cytochromes we have extended our investigations of the oxidation and reduction of Rhodospirillum rubrum cytochrome c₂. We have utilized the oxidant, oxidized azurin, and the reductants SO₂^?, S₂O₄^2?, sodium ascorbate, and reduced azurin. The results of these studies demonstrate that, as found previously with the iron hexacyanides, electron transfer apparently takes place at the exposed heme edge. Furthermore, we report studies on the reduction of ferricytochrome c₂ from Rhodopseudomonas sphaeroides, Rhodopseudomonas capsulata, Rhodomicrobium vannielii, and Rhodopseudomonas palustris by potassium ferrocyanide. Based on the amino acid sequence homology between the various cytochromes c₂ and presumed structural homology, the observed rates of electron transport are analyzed in terms of the structure in the region of the exposed heme edge.     

Purification and characterization of two new c-type cytochromes involved in Fe2+ oxidation from Thiobacillus ferrooxidans

Abstract Two new c -type cytochromes have been purified from cell membranes of the acidophilic Thiobacillus ferrooxidans . In contrast to a soluble cytochrome c with molecular mass of 14 kDa reported earlier, a membrane-bound cytochrome c with a mass of 21 kDa was solubilized with octylthioglucoside and purified to homogeneity. In addition, a high molecular mass c -type cytochrome (68 kDa) was also solubilized and purified using Triton X-100 as a detergent. Both acid-stable species are partially released during osmotic shock and chloroform treatment of the bacteria; they are integral components in the respiratory chain donating electrons to the terminal cytochrome oxidase.     

Microbiological oxidation of synthetic chalcocite and covellite by Thiobacillus ferrooxidans.

The microbiological oxidation of synthetic chalcocite and covellite has been investigated using an adapted strain of Thiobacillus ferrooxidans. Biodegradation of chalcocite was found to be 90 to 100% and that of covellite 45 to 60%. Optimum conditions for the oxidation of chalcocite were: pH, 1.7 to 2.3; temperature, 35 C; and ferric iron concentration in the range of 0.004 to 0.01 M. For covellite, the optimum conditions were: pH 2.3; temperature, 35 C; and ferric iron concentration in the range of 0.004 to 0.02 M. The energies of activation were determined to be 16.3 kcal (ca. 6.8 X 10(4) J) per mol and 11.7 kcal (ca. 4.8 X 10(4) J) per mol for chalcocite and covellite, respectively.