title: Application of β-glucuronidase (GusA) as an effective reporter for extremely acidophilic Acidithiobacillus ferrooxidans.
abstract: Acidithiobacillus ferrooxidans is a model organism for investigating metal sulfide bioleaching. The regulatory mechanism of gene expression by metabolizing various substrates is critical for understanding its role in bioleaching processes. However, no reporter has been successfully employed to study gene expression in A. ferrooxidans to date. In this study, a sensitive and robust reporter system based on β-glucuronidase (GusA) was described for feasible application in A. ferrooxidans. A set of vectors, which contained the transcriptional and translational fusions of gusA, were constructed and employed to analyze promoter activity and efficiency of translation initiation in A. ferrooxidans. Ptac and P2811 were screened out from ten tested promoters and could be used as strong promoters for gene overexpression in A. ferrooxidans. Among the four translational fusions of gusA with different start codons, ATG was most active, followed by TTG and GTG, while CTG showed the least activity. The transcriptional inhibition effect of an IclR-like transcription factor was also observed on its own encoding gene AFE_1668 as well as its neighboring AFE_1667. In addition, the specific chromogenic reaction of GusA could be detected and visualized by colonies of A. ferrooxidans containing gusA expression plasmids. Generally, the established GusA reporter system would be applied not only for quantitative analysis of promoter strength and its transcriptional regulation but also for qualitative colony screening in A. ferrooxidans in the future.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong Province, 250100, People's Republic of China.|Department of Gynecologic Oncology, Laboratory of Microbial Ecosystem, Anhui Provincial Cancer Hospital, Hefei, 230031, People's Republic of China.
authors.forename: Huiyan
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong Province, 250100, People's Republic of China.
authors.forename: Liangyan
authors.initials: L
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong Province, 250100, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wen
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong Province, 250100, People's Republic of China.
authors.forename: Jianqun
authors.initials: J
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong Province, 250100, People's Republic of China. liuxiangmei@sdu.edu.cn.
authors.forename: Xiangmei
authors.identifier: http://orcid.org/0000-0002-2908-184X
authors.initials: X
authors.lastname: Liu
mesh_terms: D042763:Acidithiobacillus / Q000235:genetics*; D000143:Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018387:Codon, Initiator; D004274:DNA, Recombinant; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: D000143:Acids; D001426:Bacterial Proteins; D018387:Codon, Initiator; D004274:DNA, Recombinant; D005966:Glucuronidase
keywords: Acidithiobacillus ferrooxidans; Promoter activity; Reporter gene; Start codon; β-Glucuronidase