State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China.
Microbiol Res. 2013 Oct 1;168(8):469-76. doi: 10.1016/j.micres.2013.04.003. Epub 2013 Apr 29.
The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38±0.64×10(-5). The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75±2.7% and 5-6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45mM of NaAsO2. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.
包括嗜酸硫杆菌（Acidithiobacillus caldus）在内的生物采矿细菌的遗传改良可以促进含硫矿物的生物浸出过程。然而，可用于嗜酸硫杆菌的有效载体非常稀缺，仅限于相对较大的广宿主范围 IncQ 质粒。在这项研究中，基于不属于 IncQ、IncW 或 IncP 组的质粒 pBBR1MCS-2，构建了一组小型可移动质粒载体（pBBR1MCS-6、pMSD1 和 pMSD2）。通过插入卡那霉素抗性报告基因，确定了 5.8kb 的 pMSD2 上 tac 启动子的功能。重组质粒 pMSD2-Km 通过接合成功转入嗜酸硫杆菌 MTH-04，转化率为 1.38±0.64×10(-5)。pMSD2-Km 在嗜酸硫杆菌 MTH-04 中的稳定性和质粒拷贝数分别为 75±2.7%和每个细胞 5-6 个拷贝。通过将 arsABC 操纵子插入 pMSD2，构建了抗砷重组质粒 pMSD2-As，并通过接合转入嗜酸硫杆菌 MTH-04。含有 pMSD2-As 的嗜酸硫杆菌 MTH-04 的砷耐受性明显提高到 45mM 的 NaAsO2。这些载体可应用于嗜酸硫杆菌及其他生物浸出细菌的遗传改良。
