Anti-mercury Vector Construction And Expression Of Thiobacillus Caldus

The strain of Acidithiobacillus caldus was firstly isolated in 1994. It is a gram-negative, extremely acidophilic obligately autotrophic bacterium. It has been used industrially in metal leaching from mineral ores and in microbial desulfurization of coal in combination with Acidithiobacillus ferrooxidans. However, the drawbacks of slow growth rate, low cell yield, and being sensitive to heavy metals have limited its further use. So it is necessary to improve Acidithiobacillus caldus, using the method of recombinant gene technology.Based on the plasmid pJRD215 that belongs to the group of wide-host-range IncQ plasmids, pTMJ212 and pTMJ213 which carrying mercury resistant gene from acidphilic chemoautotroph bacteria of Thiobacillus ferrooxidans was constructed by us. The mercry-resistant plasmids were transferred into E.coli SM10, and the MIC value of E.coli SM10 harboring pTMJ212, pTMJ213 was improved from 5 μg/ml to 22.5 μg/ml and 12.5 μg/ml, respectively.Then, the pTMJ212 of higher MIC value was transferred into A. caldus througing conjungation using the donor of E.coli SM10 that contained chromosome integrated plasmid RP4 of IncQ group. The successful gene transfer to A. caldus was confirmed using the following experiments. The transformant of A. caldus was used as the donor, and the broad-host-range IncQ group vector of pTMJ212 was mobilized to E.coli C600 that contained the IncQ group plasmid RP4. The plasmids of Km and Sm resistant E.coli C600 were extracted, and compared with the plsmids from E.coli SM10. It was verified that the mercury-resistant plasmid of pTMJ212 was successfully transferred to A. caldus.The cell growth of wild type A. caldus and A.caldus (pTMJ212) in various concentrations of HgCl₂ were compared. Cell concentration was measured in terms of protein concentration. The cell growth of mecury resistant A. caldus (pTMJ212) under HgCl₂ inhibition was clearly increased. After cultivation of A. caldus (pTMJ212) for about 50 passages in Starky-S⁰ medium without any selective pressure, 75 % of the A. caldus cells still had the mecury-resistant plasmid. It was...