Recombinant expression using the tetrathionate hydrolase promoter in Acidithiobacillus ferrooxidans.

In the iron- and sulfur-oxidizing acidophilic chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, tetrathionate hydrolase gene (Af-tth) is highly expressed during tetrathionate growth. The expression levels of Af-tth were specifically determined by quantitative reverse transcription-polymerase chain reaction and the expression ratios of S/Fe and SO/Fe were found to be 68 ± 21 and 181 ± 5, respectively. The transcriptional start site was identified by primer extension.

Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.

Identification of a gene encoding a novel thiosulfate:quinone oxidoreductase in marine Acidithiobacillus sp. strain SH.

Sulfur-oxidizing bacteria that are halophilic and acidophilic have gained interest because of their potential use in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH, which was previously identified as Acidithiobacillus thiooxidans based on its 16S rRNA gene sequence, is a chemolithoautotrophic marine bacterium exhibiting sodium chloride-stimulated thiosulfate-oxidizing activities.

Draft Genome Sequence of sp. Strain SH, a Marine Acidophilic Sulfur-Oxidizing Bacterium.

We announce here the genome sequence of a marine acidophilic sulfur-oxidizing bacterium, sp. strain SH. The bacterium has potential for use in bioleaching of sulfide ores from seawater and contains a noble gene for thiosulfate quinone oxidoreductase in addition to specific genes for the oxidation of reduced inorganic sulfur compounds.

Characterization of tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH.

Tetrathionate hydrolase (4THase), a key enzyme of the S-intermediate (S4I) pathway, was partially purified from marine acidophilic bacterium, Acidithiobacillus thiooxidans strain SH, and the gene encoding this enzyme (SH-tth) was identified. SH-Tth is a homodimer with a molecular mass of 97 ± 3 kDa, and contains a subunit 52 kDa in size. Enzyme activity was stimulated in the presence of 1 M NaCl, and showed the maximum at pH 3.

Characterization of a novel thiosulfate dehydrogenase from a marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH.

A marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH, was isolated to develop a bioleaching process for NaCl-containing sulfide minerals. Because the sulfur moiety of sulfide minerals is metabolized to sulfate via thiosulfate as an intermediate, we purified and characterized the thiosulfate dehydrogenase (TSD) from strain SH. The enzyme had an apparent molecular mass of 44 kDa and was purified 71-fold from the solubilized membrane fraction.

Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan.

Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51 mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis.

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity.

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium.

Crystallization and preliminary X-ray diffraction analysis of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity.

Tetrathionate-forming thiosulfate dehydrogenase from the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans.

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells.

Reconsideration of the Iwasaki-Waggener iterative perturbation method for reconstructing high-energy X-ray spectra.

We have reviewed applicable ranges for attenuating media and off-axis distances regarding the high-energy X-ray spectra reconstructed via the Iwasaki-Waggener iterative perturbation method for 4-20 MV X-ray beams. Sets of in-air relative transmission data used for reconstruction of spectra were calculated for low- and high-Z attenuators (acrylic and lead, respectively) by use of a functional spectral formula. More accurate sets of spectra could be reconstructed by dividing the off-axis distances of R = 0-20 cm into two series of R = 0-10 cm and R = 10-20 cm, and by taking into account the radiation attenuation and scatter in the buildup cap of the dosimeter.

A convolution/superposition method using primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance: comparison of calculated and measured 10-MV X-ray doses in thorax-like phantoms.

We performed experimental studies on the convolution/superposition method reported in the former companion paper (Iwasaki in Radiol Phys Technol 4, 2011) using 10-MV X-ray beams from open-jaw-collimated fields. The method uses primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance. We made a comparison of calculations and measurements in water phantoms and thorax-like phantoms with respect to percentage depth dose curves, tissue-phantom ratio curves, and dose profiles.

A convolution/superposition method using primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance: a theoretical study on 10-MV X-ray dose calculations in thorax-like phantoms.

A convolution/superposition method is proposed for use with primary and scatter dose kernels formed for energy bins of X-ray spectra reconstructed as a function of off-axis distance. It should be noted that the number of energy bins is usually about ten, and that the reconstructed X-ray spectra can reasonably be applied to media with a wide range of effective Z numbers, ranging from water to lead. The study was carried out for 10-MV X-ray doses in water and thorax-like phantoms with the use of open-jaw-collimated fields.

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans.

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis.

Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding.

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form.

Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene.

Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A.

Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans.

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively.

Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans.

Tetrathionate is one of the most important intermediates in dissimilatory sulfur oxidation and can itself be utilized as a sole energy source by some sulfur-oxidizing microorganisms. Tetrathionate hydrolase (4THase) plays a significant role in tetrathionate oxidation and should catalyze the initial step in the oxidative dissimilation when sulfur-oxidizing bacteria are grown on tetrathionate. 4THase activity was detected in tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells but not in iron-grown cells.

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.

Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine.

Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis.

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.

Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon.

ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP.

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.

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.

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.