Obligate Autotrophic Extremely Acidophilic Sulfur Bacteria Highly Efficient Expression Vector And,

The strain of Acidithiobacillus spreading in sulfide deposits, acid mine water and soil is a gram-negative, extremely acidophilic obligately autotrophic bacterium. Of the importance of sulfur cycle in nature, it has been used industrially in metal leaching from mineral ores and microbial desulphurization of coal in combination with other Acidibacillus. However, the drawbacks of slow growth rate, low cell yield, and being sensitive to heavy metals have limited its further use. Along with the development of Acidithiobacillus genetics and molecular biology, the available expressing vectors can't meet to the request of Acidithiobacillus genetic engineered strain. So, now it is necessary in the improvement of Acidithiobacillus, using the method of recombinant gene technology.Based on the plasmid pBBR1MCS-2 that belongs to the group of wide-host-range IncR plasmids, with high copy numbers, multiple cloning site, low molecular weight, using the streptomycin resistant gene from E. coli and the green fluorescence protein gene from jellyfish Aequorea Victoria respectively as report genes, under the controls of the strong promoter of P_tac, pBBR-tac-Sm and pBBR-tac-EGFP were constructed. The recombinant expressing vectors were transferred into E .coli JM109, the MIC value of E .coli JM109 harboring pBBR-tac-Sm was improved to 12 μg/ml; the fluorescence intensity of E. coli JM109 harboring pBBR-tac-EGFP were improved by 8 times compared to that E. coli JM109 and 3.26 times compared to that E. coli JM109 harboring control plasmid pJRD-EGFP, respectively.Then, the pBBR-tac-Sm was transferred into different strains of Acidithiobacillus through conjugation using the donor of E. coli SM10 that contained the integrated plasmid RP₄ of IncP group in the chromosome. The successful gene transfers to Acidithiobacillus were confirmed using the reverse experiments. The transforming of Acidithiobacillus was used as the donor, and the broad-host-range IncR group vector of pBBR-tac-Sm was mobilized to E. coli C600 that contained the IncP group plasmid RP4. And the plasmids purification from the reverse transconjugants is identity to the plasmids from E. coli SM10. It was verified that the streptomycin-resistant plasmid of pBBR-tac-Sm was successfully transferred to Acidithiobacillus.The growth rate of Acidithiobacillus (pBBR-tac-Sm) in various concentrations of streptomycin was compared. The Acidithiobacillus (pBBR-tac-Sm) had the optional growth rate compared to Acidithiobacillus (pMMB6) and Acidithiobacillus (pJRD215) controls and the value of MIC was up to 8.0 mg/ml. After cultivation of Acidithiobacillus (pBBR-tac-Sm) for about 50 generations in Starky-S° medium without any selective pressure, 63% of the Acidithiobacillus cells still had the streptomycin-resistant plasmid. It was concluded that the streptomycin-resistant gene had the highest expressing level in the new controlled plasmid of pBBR-tac-Sm was stable in Acidithiobacillus.Therefore, in this research a new expressing vector with abroad-host-range and high-efficiency-expression of foreign gene which can conjugate between E. coli SM10 and Acidithiobacillus was constructed. It provides a more suitable efficient expressing vector in the genetic modification of Acidithiobacillus applied in bioleaching.