title: Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus.
abstract: 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.
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China.
authors: Jianzhou
authors: J
authors: Meng
authors: Huiyan
authors: H
authors: Wang
authors: Xiangmei
authors: X
authors: Liu
authors: Jianqun
authors: J
authors: Lin
authors: Xin
authors: X
authors: Pang
authors: Jianqiang
authors: J
authors: Lin
mesh_terms: D042763:Acidithiobacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D003227:Conjugation, Genetic; D024881:Drug Resistance, Bacterial / Q000235:genetics; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins
keywords: Acidithiobacillus caldus; Arsenic tolerance; Broad-host-range plasmid; Plasmid copy number; tac promoter