Transpositional Mutagenesis of Thiobacillus novellus and Thiobacillus versutus

Transpositional mutagenesis of Thiobacillus novellus by Tn501 was achieved by means of the incompatibility of IncP plasmids. Tn501 insertion caused three types of mutant phenotypes: isoleucine auxotrophy, lysine auxotrophy, and a reduced ability to oxidize reduced sulfur compounds and to fix CO₂. Oxidation rates for elemental sulfur (S⁰), thiosulfate (S₂O₃²⁻), and tetrathionate (S₄O₆²⁻) in mutants of the latter type were reduced relative to those of the nonmutant control strain. Incorporation of labeled bicarbonate (H¹⁴CO₃⁻) was also significantly impaired. Although suicide vehicles were not useful for the introduction of transposons into T. novellus, this method was effective for the Tn1721-induced mutagenesis of Thiobacillus versutus. Tn1721 insertions resulted in the loss of the natural resistance of T. versutus to arsenate and gentamicin and in auxotrophies for isoleucine-valine, arginine, phenylalanine, valine, and panthothenate. Transpositional mutagenesis by either method should prove to be a useful tool for further study of these and other members of the genus Thiobacillus.     

Direct zinc sulphide bioleaching by Thiobacillus ferrooxidans and Thiobacillus thiooxidans

Summary Direct bioleaching (no iron(II) present) by Thiobacillus ferrooxidans mainly occurs on the surface of the very insoluble sulphides but is more important in solution when the sulphides are more soluble. In this case, Thiobacillus thiooxidans, normally not able to leach directly insoluble sulphides, has an effective leaching action.     

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.     

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).     

Thiobacillus ferrooxidans pili

The surface structures of Thiobacillus ferrooxidans were studied. When growing on a medium containing elemental sulphur, the cells possess peritrichously located filaments (piles) whose diameter varies from 4.5 to 7.0 nm and length, from 0.7 to 3.0 mcm. The cells of T. ferrooxidans do not have piles on a medium with ferrous iron. The physiological role of these structures for thiobacilli is discussed.     

Porin from Thiobacillus versutus

The porin of Thiobacillus versutus IFO 14567 was isolated by extraction of cell-envelopes with sodium dodecyl sulfate. It exhibited strong porin-activity after reconstitution into artificial lipid bilayer membranes. The diameter of the pore was determined as 1.6 nm, with a weak selectivity for cations being observed. The porin migrated as a single band (Mr 35 kDa) on SDS-polyacrylamide gel-electrophoresis after heating (100 degrees C, 5 min). The porin oligomer was not sensitive towards EDTA. Analytical ultracentrifugation studies demonstrated the native oligomer to be a trimer.     

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。     

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.     

Leaching Heavy Metals from Contaminated Soil by Using Thiobacillus ferrooxidans or Thiobacillus thiooxidans

Iron- and sulfur-oxidizing bacteria identified as Thiobacillus ferrooxidans and T. thiooxidans were successfully enriched from various soil samples contaminated with heavy metals and organic compounds. Depending on the growth medium, the soil sample, and the type of contaminant, the indigenous isolates solubilized > 50% of most of the heavy metals present in the solid sample (As, Cd, Co, Cr, Cu, Ni, V, Zn, B, Be). Leaching with T. ferrooxidans strains resulted in total extraction of Cd, Co, Cu, and Ni. With sulfur-oxidizing bacteria > 80% of Cd, Co, Cu, and Zn was mobilized from rainwater sludge. Pb and Ba were not detected in the leachate, given the insolubility of their sulfate compounds. An increase in pulp density up to 20%, indicating 6.6% total organic carbon in the soil and rubble leach experiment (sample 557), did not inhibit the growth of the indigenous T. ferrooxidans strain. In view of these results, bioleaching appears to have some potential for remediation of heavy metal contaminated soils.     

Enzymes of Intermediary Carbohydrate Metabolism in the Obligate Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus

Levels of enzymes operative in the Embden-Meyerhof-Parnas (glycolytic) pathway, pentose phosphate cycle, citric acid cycle, and certain other phases of intermediary carbohydrate metabolism have been compared in Thiobacillus thioparus and T. neapolitanus. All enzymes of the glycolytic pathway except phosphofructokinase were demonstrated in both organisms. There were some striking quantitative differences between the two organisms with respect to the activities of the individual enzymes of the glycolytic pathway and the citric acid cycle. Qualitative differences were also found: the isocitrate dehydrogenase activity of T. thioparus is strictly nicotinamide adenine dinucleotide phosphate (NADP)-dependent, whereas that of T. neapolitanus is primarily nicotinamide adenine dinucleotide-dependent, activity with NADP being low; the glucose-6-phosphate dehydrogenase of T. thioparus is particulate, whereas that of T. neapolitanus is partly soluble and partly particulate; the 6-phosphogluconate dehydrogenase of T. thioparus is soluble, that of T. neapolitanus is partly soluble and partly particulate. All enzymes which function in the carbon reduction cycle were present at very high levels. In contrast, enzymes which operate exclusively in cycles other than the carbon reduction cycle were present at low levels. Of the enzymes not operative in the carbon reduction cycle that were examined, isocitric dehydrogenase had the highest specific activity. Both organisms possessed reduced nicotinamide adenine dinucleotide dehydrogenase activity. The qualitative and quantitative aspects of the data are discussed in relation to possible biochemical explanations of obligate autotrophy.     

Intergeneric conjugation in Thiobacillus versutus

D.L. READ, L.M. TOTH AND K. McCANN. 1992. In plate matings with Escherichia coli HB101/pUW965: Tn5 (Km^R) Thiobacillus versutus reacted as an efficient recipient, producing 10^-2 to 10^-3 kanamycin resistant (Km^R) T. versutus exconjugants per donor cell. Analysis of agarose gels of plasmid DNA extracted from the exconjugants confirmed that the suicide vector pUW964 did not persist in the recipient, implying that the kanamycin resistance of the exconjugants is based on effective transposition of Tn5 in T. versutus as well as function of the E. coli kanamycin gene. Transfer was equally efficient when a nalidixate-resistant T. versutus mutant was used as recipient. Hybridization evidence for the presence of Tn5 was consistently negative. The significance of this anomalous result is discussed.     

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.     

Sulfur metabolism in Thiobacillus denitrificans

The relatively high specific sulfite reductase activity of 25 mU/mg protein was found in extracts from Thiobacillus denitrificans. The absorption spectrum of the partially purified enzyme was similar to the siroheme containing sulfite reductases from other sources. It is suggested that the T. denitrificans sulfite reductase may function during the oxidation of reduced sulfur compounds.