The Characterization And Gene Knockout Of Rhodanese In Acidithiobacillus Caldus

Acidithiobacillus caldus is a moderately thermophilic, acidophilic, chemolithotrophic, Gram-negative, sulfur-oxidizing bacterium. It gains energy for growth by oxidizing reduced inorganic sulfur compounds. A. caldus plays an important role in industrial bioleaching operations as a sulfur consumer. But there is little progress in sulfur metabolism in A. caldus owing to the adverse factors, such as the poor growth rate, sensitive to organic matter, extremely acidic environment, the complexity of sulfur oxidation system, etc.Rhodaneses belong to the sulfurtransferase family, which are found in organisms from all three domains of life and involved in all different cellular processes. The physiological functions of rhodaneses are still widely debated due to that the physiological substrates remain unclear, although rhodaneses are found for more than seventy years. There are four annotated rhodanese genes in A. caldus MTH-04genome. The gene cluster including rhd2475gene was hypothesized to participate in sulfur metabolism in A. caldus KU and Acidithiobacillus ferrooxidans ATCC23270using OMICS technologies.To better understand the functions in sulfur metabolism in A. caldus, rhodanese was heterologously expressed and purified, and then the enzymatic property was tested in this thesis. Meanwhile, the role of the rhodanese was investigated using gene knockout technology. Firstly, the feasibility analysis of the gene deletion of the rhodanese was performed by promoter analyses and reverse transcription PCRs, and then the gene replacement suicide plasmid pRE-rhd was constructed. The suicide plasmid was transferred into A. caldus by conjugation, and the transconjugants were screened on Starkey-Na2S2O3solid medium supplemented with kanamycin. The results showed that all fifty seven colonies were single-recombinants. One possible reason is that rhodanese gene is an essential gene and the deletion mutations are lethal. To keep deletion mutations alive, a plasmid pIS-rhd expressing rhodanese was obtained and expresses the meganuclease I-Sce Ⅰ as well. The recognition site for I-Sce Ⅰ is introduced into the markerless gene knockout suicide plasmid pML-rhd which derived from the suicide plasmid pRE-rhd. The single-recombinants in which plasmid pML-rhd are integrated into the chromosome were obtained through kanamycin screening by conjugation. After plasmid pIS-rhd was introduced into the single-recombinant cells, Ⅰ-Sce Ⅰ recognized the18-bp specific sequence on the suicide plasmid integrated in the chromosome, and generates a double-stranded DNA break. The double-stranded break stimulated the host DNA repair system on the basis of homologous recombination. The recombination frequency increases and the markerless deletion mutation was obtained with the help of Ⅰ-Sce Ⅰ. Three mutations strains and seven wild strains were obtained from150colonies, and the method for the deletion of essential genes was studied in A. caldus.