The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world. This bacterium oxidizes elemental sulfur and other reduced inorganic sulfur compounds as the sole source of energy. The purpose of this study was to purify and characterize the tetrathionate hydrolase of A. caldus. The enzyme was purified 16.7-fold by one step chromatography using a SP Sepharose column. The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions. Its homogeneity was confirmed by N-terminal amino acid sequencing. Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers). The purified enzyme had optimum activity at pH 3.0 and 40°C and an isoelectric point of 9.8. The periplasmic localization of the enzyme was determined by differential fractionation of A. caldus cells. Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP-HPLC analysis. The activity of the purified enzyme was drastically enhanced by divalent metal ions.

The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU. The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers. The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically. In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T. caldus KU. N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite. The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism. On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T. caldus KU.Thiobacillus caldus KU (5) is a moderately thermophilic acidophile found in environments such as coal spoil heaps (17), where the oxidative dissolution of sulfide minerals occurs. This bacterium obtains its carbon by reductive fixation of atmospheric CO 2 . T. caldus is capable of oxidizing a wide range of reduced sulfur compounds, but it is incapable of oxidizing ferrous iron or pyrite. One of the products of the oxidation of reduced sulfur compounds is H 2 SO 4 , and this bacterium is able to live in acidic environments, down to pH 1.One biotechnological application of acidophilic bacteria is the biooxidation of refractory sulfidic ores for the enhanced recovery of gold (13,14). The gold is often associated with the iron sulfides pyrite (FeS 2 ) and arsenopyrite (FeAsS) as fine particles trapped within the mineral matrix. During the biooxidation process, the iron sulfides are oxidized to soluble ferric iron and sulfate, liberating the gold particles. It has been found that T. caldus is the primary sulfur oxidizer enriched from pilot scale bioleaching reactors operating at temperatures above 40ЊC (4). In a different pilot scale study of reactors operating between 45 and 50ЊC, T. caldus makes up approximately 10% of the total bacterial population (1).Recent studies regarding the sulfur metabolism by bacteria of the genus Thiobacillus have focused on two acidophilic mesophilic species, T. ferrooxidans and T. acidophilus, and a moderately thermophilic neutrophile, T. tepidarius. It has been proposed that T. acidophilus is a suitable organism for use as a model of sulfur oxidation for thiobacilli (24)....

We investigated the potential role of the three strains ofThiobacillus caldus (KU, BC13, and C-SH12) in arsenopyrite leaching in combination with a moderately thermophilic iron oxidizer,Sulfobacillus thermosulfidooxidans. Pure cultures ofT. caldus and S. thermosulfidooxidans were used as well as defined mixed cultures. By measuring released iron, tetrathionate, and sulfur concentrations, we found that the presence ofT. caldus KU and BC13 in the defined mixed culture lowered the concentration of sulfur, and levels of tetrathionate were comparable to or lower than those in the presence of S. thermosulfidooxidans. This suggests that T. caldusgrows on the sulfur compounds that build up during leaching, increasing the arsenopyrite-leaching efficiency. This result was similar to leaching arsenopyrite with a pure culture of S. thermosulfidooxidans in the presence of yeast extract. Therefore, three possible roles of T. caldus in the leaching environment can be hypothesized: to remove the buildup of solid sulfur that can cause an inhibitory layer on the surface of the mineral, to aid heterotrophic and mixotrophic growth by the release of organic chemicals, and to solubilize solid sulfur by the production of surface-active agents. The results showed that T. caldus KU was the most efficient at leaching arsenopyrite under the conditions tested, followed by BC13, and finally C-SH12.

The rates of sulfate reduction, methanogenesis, and methane loss were measured in saltmarsh sediment at monthly intervals. In addition, dissolved methane and sulfate concentrations together with pS2and pH were determined. Methane formation from carbon dioxide, but not from acetate, was detected within the same horizon of sediment where sulfate reduction was most active. Sulfate reduction was about three orders of magnitude greater than annual methanogenesis. The two processes were not separated either spatially or temporally, but occurred within the same layer of sediment at the same time of the year. Their coexistence did not seem to be the result of sulfate-depleted microenvironments within which methanogenesis could occur, but the methanogenic bacteria persisted at very low rates of activity within the same environment as the sulfate reducers. Sulfate reduction and methanogenesis are microbial processes of particular importance in anaerobic environments as they are major terminal oxidation steps in the flow of carbon and electrons. Cappenberg (8) demonstrated that the two processes were spatially separated in freshwater sediment, and Reeburgh and Heggie (21), in a review of the available data, reported that in marine sediments methane production occurred only at depths where sulfate reduction was limited by a depleted supply of available sulfate. Winfrey and Zeikus (26) showed that sulfate additions to freshwater sediment inhibited methanogenesis and hypothesized that this was possibly due to competition between the sulfatereducing and methanogenic bacteria for a common substrate. Laboratory studies by Abram and Nedwell (2, 3) confirmed that methanogens were outcompeted by sulfate-reducing bacteria for the limited amount of hydrogen produced in sediments, and suggested that this competition was a possible explanation for the apparent inhibition of methanogenesis in the presence of sulfate reduction. The present work was undertaken to investigate in greater detail the relationship between sulfate reduction and methanogenesis in East Coast, United Kingdom, saltmarsh sediment.

The chromosomally mediated penicillinase present in three strains of Escherichia coli K-12 has been purified and characterized. Two of the strains carried the ampA gene and the third the wild-type allele. The purification involves release of the enzyme by spheroplast formation, dialysis, chromatography on sulfoethyl cellulose, and chromatography on hydroxylapatite. Enzyme from the two mutants appeared homogeneous in polyacrylamide gel electrophoresis. Enzyme from the wild-type strain gave two bands. Immunologically, the enzymes from all three strains were identical. Ultracentrifugation gave a homogeneous peak with a sedimentation coefficient of 3.45. Gel filtration gave an estimated molecular weight of29,000. The Nterminal amino acid residue was found to be alanine. Complete amino acid analysis showed a lack of cysteine. Ultraviolet spectra were recorded at three different pH values. The extinction coefficient at 280 nm is 21.0 for a 1% solution at pH 6.8. The optimal pH is 7.3. With enzyme from one of the resistant mutants, the following Km and turnover number values were obtained: for penicillin G, 12 ;uM and 2,080; for D-MATERIALS AND METHODS Bacterial strains. All strains used were E. coli K-12. Strain Gllal which carries the amp Al allele is a spontaneous mutant obtained from the wild-type strain Gll (10). The ampAl allele was transduced into strain D2, which afterwards was mutated to the highly ampicillin-resistant strain D3 (5). A second mutation step induced streptomycin resistance in D3, thus producing strain D31 (5). Strains Gllal and D2 both form single cell colonies on plates containing D-ampicillin concentrations of 15 to 20,g/ml, whereas strain D31 can form single-cell colonies on plates with D-ampicillin concentrations of 75 to 100 ,Ag/ml (for resistance determinations, see references 6 and 24). Media and growth conditions. The growth medium 218