Phosphoenolpyruvate carboxylase of Thiobacillus thioparus. I. General properties.

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxylase (phosphorylating), EC 4.1.1.31) was purified 19-fold from the obligate chemoautotroph, Thiobacillus thioparus. Michaelis constants for the substrates were found to be 0.44 mM for phosphoenolpyruvate, 0.89 mM for bicarbonate, and 0.37 mM for magnesium, using Tris-HC1, pH 7.3. 1-Aspartate, 1-malate, and orthophosphate were found to be inhibitors of enzyme activity, while acetyl CoA, FDP, GTP, and CDP had no effect. Dioxane greatly stimulated enzyme activity.     

氧化硫硫杆菌的研究进展

综述了氧化硫硫杆菌(Thiobacillus thiooxidans,T.t)培养方法和应用方面的研究进展。     

Purification and some properties of cytochrome c-552 from a sulfur-oxidizing bacterium, Thiobacillus thiooxidans.

A soluble cytochrome c-552 from Thiobacillus thiooxidans was highly purified and its physico-chemical properteis were studied. The absorption maxima were at 552,523,418 nm in the reduced from and at 412 nm in the oxidized form. The pyridine hemochrome spectrum was the same as that of other cytochromes c. The molecular weight, estimated by the gel filtration method, was found to be 12,600. The isoelectric point was determined to be 9.2-9.3 by the electrofocusing technique. The standard oxidation-reduction potential of this cytochrome was +0.247 V.     

Phospholipids of Thiobacillus thiooxidans

Cells and spent growth media from sulfur- and thiosulfate-grown cultures of Thiobacillus thiooxidans were analyzed. The phosphatides were examined by thinlayer chromatography, and the products of their hydrolysis by hydrochloric acid and methanolic potassium hydroxide were separated by paper chromatography. The phospholipids in both cells and spent growth media were identified as phosphatidyl ethanolamine, phosphatidyl-N-monomethylethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. These comprised about 97% of the total lipid phosphorus. Lyso-phosphatidyl-N-monomethylethanolamine and lysophosphatidyl glycerol accounted for the remaining 3%. The percentage of the total lipid phosphorus accounted for by each phospholipid depended on the age of the culture.     

Fine Structure of Thiobacillus thiooxidans

Mahoney, Robert P. (Skidmore College, Saratoga Springs, N.Y.), and Mercedes R. Edwards. Fine structure of Thiobacillus thiooxidans. J. Bacteriol. 92: 487-495. 1966.-Thin section analysis of the chemosynthetic autotroph Thiobacillus thiooxidans revealed structures comparable to gram-negative heterotrophic bacteria. Although this species is unique in that it oxidizes elemental sulfur for energy, uses carbon dioxide as its sole source of carbon, and can withstand a pH of less than 1, thin sections revealed a profile of the cell envelope (cell wall and plasmalemma) similar to other gram-negative species which have more common physiological traits. The cell wall is composed of five layers with an overall width of approximately 200 A, and the plasmalemma appears as a conventional "unit membrane" with a width of about 85 A. Volutin granules and less-dense bodies of similar shape and size were frequently observed in close association with the nucleoplasm. The nature and function of these bodies are unknown at this time.     

Fatty Acids of Thiobacillus thiooxidans

Fatty acid spectra were made on Thiobacillus thiooxidans cultures both in the presence and absence of organic compounds. Small additions of glucose or acetate had no significant effect either on growth or fatty acid content. The addition of biotin had no stimulatory effect but did result in slight quantitative changes in the fatty acid spectrum. The predominant fatty acid was a C₁₉ cyclopropane acid.     

Extracellular Lipid of Thiobacillus thiooxidans

The extracellular lipid of Thiobacillus thiooxidans is a heterogeneous mixture of phospholipid and neutral lipid, primarily free fatty acids.     

Basis of pyruvate inhibition in Thiobacillus thiooxidans

Addition of 10(-3)m pyruvic acid to cultures of Thiobacillus thiooxidans, at pH 2.3, results in its rapid intracellular accumulation and in the cessation of sulfur oxidation, CO(2) fixation, and oxygen consumption; at pH 7.0, pyruvate neither inhibits oxygen uptake nor accumulates appreciably intracellularly. Pyruvate does not affect CO(2) fixation in cell-free extracts. The data suggest that the cells of T. thiooxidans are passively permeable to pyruvic acid at low pH. Thus entry of pyruvic acid causes accumulation of pyruvate with a concomitant decrease in intracellular pH.     

Combined degradation of covellite by Thiobacillus thiooxidans and Thiobacillus ferrooxidans

Summary In the presence of iron, which is always associated with natural sulphide ores, the percentages of copper dissolution in the bioleaching of covellite were 34 and 45 % when Thiobacillus thiooxidans and Thiobacillus ferrooxidans were used together and when an indirect bioleaching with attached bacteria was performed respectively. In the latter, the percentage of copper dissolution was still higher than the percentages obtained with pure cultures (36 % with a T. thiooxidans culture and 40 % with a T. ferrooxidans culture).     

Preservation of Thiobacillus ferroxidans and Thiobacillus thiooxidans with activity check

Cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, used in biohydrometallurgical processes of economic importance, are very difficult to preserve by conventional methods. Hence, to preserve the cultures with their activity intact, various techniques were tried, after determining their respective activity in terms of Iron Oxidation Rate (IOR) and Sulfur Oxidation Rate (SOR). Among the methods tested, along with the recommended method of serial transfer in a liquid medium, were methods such as lyophilization, storage in a liquid nitrogen and mixing with sterile, inert carriers like lignite or chalcopyrite ores. After a period check-up at 4 months and 8 months storage, it was found that out of these methods, mixing with sterile ore followed by storage at 8°C, kept both types of activities intact. The temperature of storage was observed to have a definite effect on activity, in that when the preserved cultures were stored at 8°C, the activity was retained, whereas at 28–30°C (RT) storage, the activity of all the cultures preserved by various techniques, dropped significantly.     

Acidostability of spheroplasts prepared from Thiobacillus thiooxidans

Thiobacillus thiooxidans was acidostable even in the absence of its respiratory substrate, elementary sulfur. This suggests that the acidostability of the bacterium was energy-independent. The organism was subjected to osmotic shock with 0.75 M sucrose at 0°C and then treated with snail intestinal juice in the presence of 0.3 M sucrose. The decrease in the optical density of the sample thus prepared on dilution with deionized water and electron microscopic observation of the sample showed that spheroplasts were formed from the bacterium by this procedure. Spheroplasts were able to respire sulfur and their respiratory activity was acidostable. Spheroplasts, when treated with Nagase, proteolytic enzyme, lost their acidostability, and some protein components disappeared from the membrane fraction. This suggests that the acidostability of the bacterium may be related to protein components of the membrane.     

Studies on the metabolism of a sulfur-oxidizing bacterium IV.1 Growth and oxidation of sulfur compounds in Thiobacillus thiooxidans

By immersing a few small cellophane bags containing BaCO³ powderin STARKEY's medium, the duration of lag phase in the growthof Thiobacillus thiooxidans is minimized and the yield of cellsis increased ten times that of the previous method. The activitiesof oxidation for sulfur and sulfite change with growth. Sulfiteis oxidized at a comparable rate to that of sulfur oxidationat pH values between 6.0 and 6.5. In the presence of cysteineor glutathione, thiosulfate can be oxidized at a pH above 5.0.At pH values below 4.5, apparent oxidation of thiosulfate andtetrathionate to sulfate is observed. This result is accountedfor by the facts that thiosulfate is decomposed to sulfur andsulfite under the acidic condition at pH values below 4.5, andthat tetrathionate is reduced to thiosulfate enzymatically.In the oxidation of tetrathionate, oxygen uptake begins aftera lag phase, the duration of which depends on the concentrationsof cells and of tetrathionate. Cysteine is oxidized to cystine.The oxidation is strongly inhibited by metal-chelating agents.The cysteine oxidizing activity is, however, quite stable andis not lost by treating cells with organic solvents, sonic oscillation,by heating or lyophilization. ¹III=References (11). ²Partly supported by a grant from the Ministry of Education.