| As the mineral resources beginning to tailing off, it is a better way to recycle the low-grade mineral resources and gangue by bioleaching. Acidithiobacillus caldus, a kind of extremely acidophilic autotrophic bacterium, is an important microbe commonly used in bioleaching and widely distributed in the sulfide deposits. Due to its slow growth rate, low cell yields and sensitivity to some heavy metals, its actual efficient in large-scale mineral processing has been restricted. In order to improve its capacity in bioleaching, the genetic modification on this strain seems necessary and urgent. Now conjugation has been proved to be a successful way to transfer foreign genes into Acidithiobacillus. However, the IncQ group plasmid vectors used in the conjugation system can't satisfy with further research, because of their large molecular weights, less MCS, and difficulty in genetic manipulations. It's necessary to construct new plasmid vectors to meet the needs for the research and application.In this article, based on the wide-host-range IncR plasmid pBBR1MCS-2, two new vectors, plasmid pMSD2 (5,833 bp) and plasmid pMSD1 (5,602 bp), were constructed. The streptomycin risitant gene was substituted for the kanamycin resisitant gene as selecting marker. Plasmid pMSD1 had no promtor and pMSD2 had a strong promoter tac. In order to validate the characteristics of the two vectors, the kanamycin resistant gene was selected as reporter gene to construct two recombinant plasmids, pMSD1-Km and pMSD2-Km. Compared with the former IncQ group plasmid pJRD215 carrying the same kanamycin resistant gene, the expression levels of the reporter gene in the three plasmids both in E. coli JM109 and A. caldus MTH-04 were detected. The results showed that the highest expressing capacity of kanamycin resistant gene was obtained in plasmid pMSD2 under the strong promotor tac. While under its own promotor, the expression of kanamycin resistant gene in plasmid pMSD1 was a bit lower than that in control plasmid pJRD215. At the same time, the comparison was also carried out on the conjugation frequency from E: coli to A. caldus and the stability in A. caldus. Conducted by plate colony counting, the conjugation frequency of plasmid pMSD2-Km was 1.38±0.64×10-5. Incubating A. caldus harboring plasmids pMSD2-Km in free-pressure medium for about 50 generations, the retention rate of plasmid pMSD2-Km was about 75±2.7%. While the conjugation frequency of control plasmid pJRD215 was 3.33±0.72×10-5, and its retention rate was 79±3.2%. The results indicated that the new constructed IncR group plasmid pMSD2 and pMSD1 could be transfered into Acidithiobacillus by conjugation and stablely existent in it, and so as to be served as expressing vecters in Acidithiobacillus strains.The plasmid copy number of plasmid pMSD2-Km and pJRD215 both in E. coli JM109 and A. caldus MTH-04 were quantified by real-time quantification PCR. In E. coli JM109, the single copy D-1-deoxyxylulose 5-phosphate synthase gene was chosen as reference gene, while the kanamycin resistant gene in plasmids was chosen as target gene. In A. caldus MTH-04, the gene sequence for the big sununit of ribulose-1, 5-biphosphate carboxylase/oxygenase was chosen as reference gene and the streptomycin resistant gene in plasmid as target gene. The whole DNA of E. coli (pMSD2-Km), E. coli (pJRD215), A. caldus (pMSD2-Km) and A. caldus (pJRD215) were extracted and conducted for real-time quantification PCR, respectively. The results showed that the plasmid copy number of pMSD2-Km and pJRD215 were about 5-6 copies(4.2%-6.3%) and 15-17 copies (4.7%-6.9% ) in E. coli JM109, while were about 5-6 copies(3.0%-5.2%) and 14-15 copies (2.3%-4.5%) in A. caldus MTH-04, respectively.Then, two recombinant arsenic resistant plasmids, pMSD1-As and pMSD2-As, were constructed by cloning the arsenic resistant gene from E. coli plasmid pUM3 into the two new plasmid pMSD1 and pMSD2. And two new arsenic resistant engineeting strains, A. caldus MTH-04 (pMSD1-As) and A. caldus MTH-04 (pMSD2-As), were obtained respectively by transferring plasmid pMSD1-As and pMSD2-As into A. caldus MTH-04 by conjugation. Compared with the former arsenic resistant plasmid pSDRA1 based on pJRD215, the ability to resist different concentrations of sodium arsenite by different engineering strains was detected. The results showed that the maximal tolerant concentration of sodium arsenite was about 40 mmol/L for control A. caldus (pSDRA1), and 40 mmol/L for the new construced A. caldus (pMSD1-As) with less cell yields. However, the new construced A. caldus (pMSD2-As) had the highest capacity on arsenic resistance and its maximal tolerant concentration of sodium arsenite was about 45 mmol/L.In this experiment, two new broad-host-range IncR plasmids pMSD1 and pMSD2 were constructed. They have improved characteristics, such as small molecular weights, high frequency of transfer into Acidithiobacillus by conjugation, high efficiency of expressing foreign genes, and stablely existent in Acidithiobacillus. The quantification of plasmid number of pMSD2 and pJRD215 both in E. coli and A. caldus strains were also carried out. At last, based on the plasmids pMSD1 and pMSD2, new engineering A. caldus strains were constructed with higher capacity to arsenic resistance. |
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